Inhibitors of protein kinases

ABSTRACT

The present invention is directed to 2-cycloalkylamino-4-arylamino-benzamide compounds of formula I pharmaceutically acceptable salts thereof which are inhibitors of syk and/or JAK kinase. The present invention is also directed to intermediates used in making such compounds, the preparation of such a compound, pharmaceutical compositions containing such a compound, methods of inhibition syk and/or JAK kinase activity, and methods to prevent or treat a number of conditions mediated at least in part by syk and/or JAK kinase activity, such as cardiovascular disease, inflammatory disease, autoimmune disease and cell proliferative disorder.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is claims the benefit of U.S. Provisional PatentApplication No. 61/120,348, filed Dec. 5, 2008; U.S. Provisional PatentApplication No. 61/120,344, filed Dec. 5, 2008; U.S. Provisional PatentApplication No. 61/045,406, filed Apr. 16, 2008; and U.S. ProvisionalPatent Application No. 61/045,417, filed Apr. 16, 2008; the entiredisclosures of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is directed to pyrimidine-5-carboxamide compounds whichact as inhibitors of Spleen tyrosine kinase (syk) and/or JAK kinases.This invention is also directed to pharmaceutical compositionscontaining the pyrimidine-5-carboxamide compounds and methods of usingthe compounds or compositions to treat a condition characterized byundesired thrombosis. The invention is also directed to methods ofmaking the compounds described herein.

2. State of the Art

Protein kinases constitute a large family of structurally relatedenzymes that are responsible for the control of a variety of signaltransduction processes within cells (see, e.g., Hardie and Hanks, TheProtein Kinase Facts Book, I and II, Academic Press, San Diego, Calif.,1995). Protein kinases are thought to have evolved from a commonancestral gene due to the conservation of their structure and catalyticfunction. Almost all kinases contain a similar 250-300 amino acidcatalytic domain. The kinases can be categorized into families by thesubstrates they phosphorylate (e.g., protein-tyrosine,protein-serine/threonine, lipids, etc.). Sequence motifs have beenidentified that generally correspond to each of these families (see,e.g., Hanks & Hunter, (1995), FASEB J. 9:576-596; Knighton et al.,(1991), Science 253:407-414; Hiles et al., (1992), Cell 70:419-429; Kunzet al., (1993), Cell 73:585-596; Garcia-Bustos et al., (1994), EMBO J.13:2352-2361).

Many diseases are associated with abnormal cellular responses triggeredby protein kinase-mediated events. These diseases include autoimmunediseases, inflammatory diseases, bone diseases, metabolic diseases,neurological and neurodegenerative diseases, cancer, cardiovasculardiseases, allergies, asthma, alzheimer's disease and hormone-relateddiseases. As a consequence, there has been substantial efforts inmedicinal chemistry to find inhibitors of protein kinases for use astherapeutic agents.

Immunoreceptor tyrosine activation motif (ITAM)-mediated signaling hasemerged as a primary event in signaling pathways responsible for humanpathologies. ITAM-mediated signaling is responsible for relayingactivation signals initiated at classical immune receptors such asT-cell receptors, B-cell receptors, Fc receptors in immune cells and atGPVI and FcγRIIa in platelets to downstream intracellular molecules suchas syk and ZAP-70 (Underhill, D. M and Goodridge, H. S., TrendsImmunol., 28:66-73, 2007).

The binding of a ligand to an ITAM-containing receptor triggerssignaling events which allows for the recruitment of proteins from afamily of nonreceptor tyrosine kinases called the Src family. Thesekinases phosphorylate tyrosine residues within the ITAM sequence, aregion with which the tandem SH2 domains on either syk or ZAP-70interact.

Syk, along with Zap-70, is a member of the syk family of proteintyrosine kinases. The interaction of syk or ZAP-70 with diphosphorylatedITAM sequences induces a conformation change in the kinases that allowsfor tyrosine phosphorylation of the kinase itself. Phosphorylated Sykfamily members activate a multitude of downstream signaling pathwayproteins which include Src homology 2 (SH2) domain containingleukocyte-specific phosphoprotein of 76 kDa (SLP-76), Linker ofActivation of T-cells (LAT) and PLC (phospholipase C)γ2.

Human pathologies attributed to dysfunctional ITAM-mediated signalinginclude autoimmune diseases such as rheumatoid arthritis, systemiclupus, multiple sclerosis, hemolytic anemia, immune-thrombocytopeniapurpura, and heparin-induced thrombocytopenia and arteriosclerosis.Interestingly, many of the above mentioned diseases are thought to occurthrough crosslinking of Fc receptors by antibodies which, via syk,activate a signaling cascade in mast, basophil and other immune cellsthat result in the release of cell mediators responsible forinflammatory reactions. The release of mediators and the production ofcytokines in IgE stimulation-dependent allergic and inflammatoryreactions from mast cells and basophiles can be controlled by inhibitingthe tyrosine kinase activity of syk (Rossi, A. B. et al., J Allergy ClinImmunol., 118:749-755, 2006). In immune-thrombocytopenia, antibody boundplatelets are cleared by the spleen by an Fc receptor/ITAM/syk-mediatedprocess (Crow, A. R. et al., Blood, 106:abstract 2165, 2005).Drug-induced thrombocytopenia, caused by heparin-platelet factor 4immune complexes that activate platelet FcγRIIa, also involve syksignaling downstream of receptor engagement (Reilly, M. P., Blood,98:2442-2447, 2001).

Platelet agonists induce inside-out integrin signaling resulting infibrinogen binding and platelet aggregation. This initiates outside-insignaling which produces further stimulation of platelets. syk isactivated during both phases of integrin signaling, and inhibition ofsyk is shown to inhibit platelet adhesion to immobilized proteins (Law,D. A. et al., Blood, 93:2645-2652, 1999). Release of arachidonic acidand serotonin and platelet aggregation induced by collagen are markedlyinhibited in platelets derived from syk deficient mouse (Poole, A. etal., EMBO J., 16:2333-2341, 1997). Thus syk inhibitors may also possessanticoagulation action.

Because of the role syk plays in Ig-induced platelet activations, it islikely to be important in arteriosclerosis and restenosis.Arteriosclerosis is a class of diseases characterized by the thickeningand hardening of the arterial walls of blood vessels. Although all bloodvessels are susceptible to this serious degenerative condition, theaorta and the coronary arteries serving the heart are most oftenaffected. Arteriosclerosis is of profound clinical importance since itcan increase the risk of heart attacks, myocardial infarctions, strokes,and aneurysms.

The traditional treatment for arteriosclerosis includes vascularrecanalization procedures for less-serious blockages and coronary bypasssurgery for major blockages. A serious shortcoming of intravascularprocedures is that, in a significant number of treated individuals, someor all of the treated vessels restenose (i.e., re-narrow). For example,restenosis of an atherosclerotic coronary artery after PTCA occurs in10-50% of patients undergoing this procedure and subsequently requireseither further angioplasty or a coronary artery bypass graft.Furthermore, restenosis of an atherosclerotic coronary artery afterstenting occurs in 10-20% of patients undergoing this procedure andsubsequently requires repeat treatments to maintain adequate blood flowthrough the affected artery. Restenosis generally occurs in a relativelybrief time period, e.g., roughly less than six months, after treatment.

While the exact hormonal and cellular processes promoting restenosishave not been determined, restenosis is thought to be due in part tomechanical injury to the walls of the blood vessels caused by theballoon catheter or other intravascular device. For example, the processof PTCA, in addition to opening the obstructed artery, also injuresresident coronary arterial smooth muscle cells (SMCs). In response tothis injury, adhering platelets, infiltrating macrophages, leukocytes,or the smooth muscle cells themselves release cell-derived growthfactors such as platelet-derived growth factor (PDGF), with subsequentproliferation and migration of medial SMCs through the internal elasticlamina to the area of the vessel intima. Further proliferation andhyperplasia of intimal SMCs and, most significantly, production of largeamounts of extracellular matrix over a period of three to six monthsresults in the filling in and narrowing of the vascular space sufficientto significantly obstruct blood flow.

In addition to the role syk plays in Ig-induced platelet activations,syk plays a very important role in collagen-mediated signaling. Theprimary adhesive protein responsible for platelet adhesion andactivation is collagen. Collagen is a filamentous protein containedwithin the fibrotic caps of atheromas which becomes exposed to bloodduring plaque rupture. Collagen functions initially by binding vonWillebrand factor which tethers platelets through binding plateletmembrane GPIb. Collagen functions secondarily by engaging the twocollagen receptors on platelets, GPVI and integrin α2β1.

GPVI exists in platelet membranes as a complex with FcRγ, an interactionrequired for the expression of GPVI. Activation of FcγRIIa on plateletsresults in platelet shape change, secretion and thrombosis. Signaling bythe GPVI/FcRγ complex is initiated by tyrosine phosphorylation of theITAM domain of FCRγ followed by the recruitment of syk. Activation ofGPVI leads to induction of multiple platelet functions including:activation of integrins α2β1 to achieve firm platelet adhesion, and GPIIb-IIIa which mediates platelet aggregation and thrombosis growth;platelet secretion, allowing for the delivery of inflammatory proteinssuch as CD40L, RANTES and TGFβ to the vessel wall; and the expression ofP-selectin which allows for the recruitment of leukocytes. Therefore, itis believed that syk inhibitors can inhibit thrombotic events mediatedby platelet adhesion, activation and aggregation.

It has been reported that the tyrosine phosphorylation of intracellularprotein (activation) induced by stimulation of a receptor for IgGantibody, FcγR, and the phagocytosis mediated by FcγR are considerablyinhibited in macrophages derived from syk deficient mouse (Crowley, M.T. et al., J. Exp. Med., 186:1027-1039, 1997). This suggests that sykhas a markedly important role in the FcγR-mediated phagocytosis ofmacrophages.

It has also been reported that an antisense oligonucleotide of syksuppresses the apoptosis inhibition of eosinophils induced by GM-CSF(Yousefi, S. et al., J. E. Med., 183:1407-1414, 1996), showing that sykis essential for the life extending signal of eosinophils caused byGM-CSF and the like. Since life extension of eosinophils is closelyrelated to the transition of diseases into a chronic state in allergicdisorders, such as asthma, syk inhibitors can also serve as therapeuticagents for chronic eosinophilic inflammation.

Syk is important for the activation of B-cells via a B-cell antigenreceptor and is involved in the phosphatidylinositol metabolism andincrease in the intracellular calcium concentration caused by theantigen receptor stimulation (Hutchcroft, J E. et al., J. Biol. Chem.,267:8613-8619, 1992; and Takata, M. et al., EMBO J., 13:1341-1349,1994). Thus, syk inhibitors may be used to control the function ofB-cells and are, therefore, expected to serve as therapeutic agents forantibody-related diseases.

Syk binds to a T-cell antigen receptor, quickly undergoes tyrosinephosphorylation through crosslinking of the receptor and synergisticallyacts upon intracellular signals mediated by Src tyrosine kinases such asLck (Couture, C. et al., Proc. Natl. Acad. Sci. USA, 91:5301-5305, 1994;and Couture, C. et al., Mol. Cell. Biol., 14:5249-5258, 1994). syk ispresent in mature T-cell populations, such as intraepithelial γδ T-cellsand naïve αβ T-cells, and has been reported to be capable ofphosphorylation of multiple components of the TCR signaling cascade(Latour, S. et. al., Mol Cell Biol., 17:4434-4441, 1997). As aconsequence, syk inhibitors may serve as agents for inhibiting cellularimmunity mediated by T-cell antigen receptor.

Recent comparative genomic hybridization studies have identified syk asanother gene important in the pathogenesis of Mantle Cell Lymphoma (MCL)(Chen, R. et al. Journal of Clinical Oncology, 2007 ASCO Annual MeetingProceedings (Post-Meeting Edition). Vol 25, No 18S (June 20 Supplement),2007: 8056). MCL represents 5-10% of all non-Hodgkins lymphomas and itis a difficult form of lymphoma to treat. It has the worst prognosisamong the B cell lymphomas with median survival of three years. It hasbeen reported that Syk is overexpressed in MCL (Rinaldi, A, et. al, Br.J. Haematol., 2006; 132:303-316) and that Syk mediates mTOR (mammaliantarget of Rapamycin) survival signals in follicular, mantel cell,Burkitt's, and diffuse large B-cell non-Hodgkin's lymphomas (Leseux, L.,et. al, Blood, 2006; 108:4156-4162).

Several lines of evidence suggest that many B-cell lymphomas depend uponB-cell receptor (BCR)-mediated survival signals. BCR signaling inducesreceptor oligomerization and phosphorylation of Igα and β immunoreceptortyrosine-based activated motifs by SRC family kinases. ITAMphosphorylation results in the recruitment and activation of syk thatinitiates downstream events and amplifies the original BCR signal. Giventhe role of tonic BCR signaling in normal B cell and syk-dependentsurvival of non-Hodgkins lymphoma cell lines in vitro (Chen, L., et. al,Blood, 2006; 108:3428-3433), syk inhibition is a promising rationaltreatment target for certain B-cell lymphomas and chronic lymphocyticleukemia (CLL) (Stefania Gobessi, Luca Laurenti, Pablo Longo, LauraCarsetti, Giuseppe Leone, Dimitar G. Efremov, Constitutive activation ofthe protein tyrosine kinase Syk in Chronic Lymphocytic Leukemia B-cells,Blood, 2007, 110, Abstract 1123). Recent data shows that administrationof a multikinase inhibitor which inhibits syk, may have significantclinical activity in CLL patients (Friedberg J W et al, Blood 2008;112(11), Abstract 3).

The oncogenic potential of the spleen tyrosine kinase (Syk) has beendescribed in a number of different settings. Clinically, Sykover-expression is reported in Mantle Cell Lymphoma (Rinaldi, A, et. al,Br. J. Haematol., 2006; 132:303-316) and the TEL-Syk fusion protein(Translocated ETS Leukemia) generated by a chromosomal translocation(t(9; 12)(q22; p12)) leads to increased Syk activity and is associatedwith myelodysplastic syndrome (Kuno, Y., et. al, Blood, 2001;97:1050-1055). Leukemia is induced in mice by adoptively transferringbone marrow cells that express human TEL-Syk (Wossning, T., JEM, 2006;203:2829-2840). Further, in mouse primary bone marrow cells,over-expression of Syk results in IL-7 independent growth in culture(Wossning, T., et. al, JEM, 2006; 203:2829-2840).

Interestingly, Syk signaling appears to be required for B-celldevelopment and survival in humans and mouse. Inducible loss of theB-cell receptor (Lam, K., et. al, Cell, 1997; 90:1073-1083) or Igα(Kraus, M., et. al, Cell, 2004; 117:787-800) results in loss ofperipheral B-cells in mice. Over-expression of the protein tyrosinephosphatase PTP-RO, which is known to negatively regulate Syk activity,inhibits proliferation and induces apoptosis in cell lines derived fromnon-Hodgkin's lymphomas (Chen, L., et. al, Blood, 2006; 108:3428-3433).Finally, B-cell lymphomas rarely exhibit loss of BCR expression, andanti-idiotype therapy rarely leads to resistance (Kuppers, R. Nat RevCancer, 2005; 5:251-262).

Engagement of the antigen-specific B cell receptor (BCR) activatesmultiple signaling pathways that ultimately regulate the cellsactivation status, promoting survival and clonal expansion. Signalingthrough the BCR is made possible by its association with two othermembers of the immunoglobulin super-family; Igα and Igβ, each bearing animmuno-tyrosine based activation motif (ITAM) (Jumaa, Hendriks et al.Annu Rev Immunol 23: 415-45 (2005). The ITAM domain is directlyphosphorylated by Src family kinases in response to BCR engagement. Thespleen tyrosine kinase (Syk) docks with and phosphorylates the ITAM, aprocess that enhances its kinase activity, resulting in Sykautophosphorylation and tyrosine phosphorylation of multiple downstreamsubstrates (Rolli, Gallwitz et al. Mol Cell 10(5): 1057-69 (2002). Thissignaling pathway is active in B cells beginning at the transition frompro- to pre-B cell stage of development, when the newly formed pre-BCRis expressed. In fact, B cell development arrests at the pro-B cellstage in Syk knockout mice (Cheng, Rowley et al. 1995; Turner, Mee etal. Nature 378(6554): 303-6 (1995). Inducible loss of the B cellreceptor (Lam, Kuhn et al. Cell 90(6): 1073-83 (1997) or Igα (Kraus,Alimzhanov et al. Cell 117(6): 787-800 (2004) results in loss ofperipheral B cells in mice. Human B cells also appear to require Syk forproliferation and survival. Over-expression of the protein tyrosinephosphatase PTP-RO, a negative regulator of Syk activity, inhibitsproliferation and induces apoptosis in cell lines derived fromnon-Hodgkin's lymphomas (NHL) (Chen, Juszczynski et al. Blood 108(10):3428-33 (2006). Knock down of Syk by siRNA in the NHL line SUDHL-4 ledto a block in the G1/S transition of the cell cycle (Gururajan, Dasu etal. J Immunol 178(1): 111-21 (2007). Together, these data suggest thatSyk signaling is required for the development, proliferation, and evensurvival of human and mouse B cells.

Conversely, the oncogenic potential of Syk has been described in anumber of different settings. Clinically, Syk over-expression isreported in Mantle Cell Lymphoma (Rinaldi, Kwee et al. Br J Haematol132(3): 303-16 (2006) and the TEL-Syk fusion protein (Translocated ETSLeukemia) generated by a chromosomal translocation (t(9; 12)(q22; p12))leads to increased Syk activity and is associated with myelodysplasticsyndrome (Kuno, Abe et al. Blood 97(4): 1050-5 (2001). Leukemia isinduced in mice by the adoptive transfer of bone marrow cells thatexpress human TEL-Syk (Wossning, Herzog et al. J Exp Med 203(13):2829-40 (2006). Further, in mouse primary bone marrow cells,over-expression of Syk results in IL-7 independent growth in culture(Wossning, Herzog et al. 2006). Consistently, Syk was reported tomediate mTOR (mammalian target of Rapamycin) survival signals infollicular, mantle cell, Burkitt's, and diffuse large B-cell NHL(Leseux, Hamdi et al. Blood 108(13): 4156-62 (2006). Additional recentstudies also suggest that Syk-dependant survival signals may play a rolein B-cell malignancies, including DLBCL, mantle cell lymphoma andfollicular lymphoma (Gururajan, Jennings et al. 2006; Irish, Czerwinskiet al. J Immunol 176(10): 5715-9 (2006). Given the role of tonic BCRsignaling in normal B cells and Syk-dependent survival of NHL cell linesin vitro, the specific inhibition of Syk may prove promising for thetreatment of certain B-cell lymphomas.

Recently, R406 (Rigel Pharmaceuticals) was reported to inhibit ITAMsignaling in response to various stimuli, including FcεR1 and BCRinduced Syk activation (Braselmann, Taylor et al. J Pharmacol Exp Ther319(3): 998-1008 (2006). Interestingly, this ATP-competitive inhibitorof Syk was also active against Flt3, cKit, and JAK kinases, but notagainst Src kinsase (Braselmann, Taylor et al. 2006). Activatingmutations to Flt3 are associated with AML and inhibition of this kinaseis currently under clinical development (Burnett and Knapper HematologyAm Soc Hematol Educ Program 2007: 429-34 (2007). Over-activation of thetyrosine kinase cKit is also associated with hematologic malignancies,and a target for cancer therapy (Heinrich, Griffith et al. Blood 96(3):925-32 (2000). Similarly, JAK3 signaling is implicated in leukemias andlymphomas, and is currently exploited as a potential therapeutic target(Heinrich, Griffith et al. 2000). Importantly, the multi-kinaseinhibitory activity of R406 attenuates BCR signaling in lymphoma celllines and primary human lymphoma samples, resulting in apoptosis of theformer (Chen, Monti et al. Blood 111(4): 2230-7 (2008). Further, a phaseII clinical trial reported favorable results by this compound inrefractory NHL and chronic lymphocytic leukemia (Friedberg J W et al,Blood 2008; 112(11), Abstract 3). Although the precise mechanism ofaction is unclear for R406, the data suggest that inhibition of kinasesthat mediate survival signaling in lymphocytes is clinically beneficial.

Additional recent studies also suggest that syk-dependant survivalsignals may play a role in B-cell malignancies, including DLBCL, mantlecell lymphoma and follicular lymphoma (see e.g., S. Linfengshen et al.Blood, February 2008; 111: 2230-2237; J. M. Irish et al. Blood, 2006;108: 3135-3142; A. Renaldi et al. Brit J. Haematology, 2006; 132:303-316; M. Guruoajan et al. J. Immunol, 2006; 176: 5715-5719; L. Laseuxet al. Blood, 2006; 108: 4156-4162.

JAK kinases (Janus Kinases) are a family of cytoplasmic protein tyrosinekinases including JAK1, JAK2, JAK3 and TYK2. The JAKs play a crucialrole in cytokine signaling. Each of the JAK kinases is selective for thereceptors of certain cytokines, though multiple JAK kinases can beaffected by particular cytokine or signaling pathways. Studies suggestthat JAK3 associates with the common cytokine receptor gamma chain (Fcγor γc) of the various cytokine receptors. JAK3 in particular selectivelybinds to receptors and is part of the cytokine signaling pathway for andactivated by IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. JAK1 interactswith, among others, the receptors for cytokines IL-2, IL-4, IL-7, IL-9and IL-21, while JAK2 interacts with, among others, the receptors forIL-9 and TNF-α. Upon the binding of certain cytokines to their receptors(e.g., IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21), receptoroligomerization occurs, resulting in the cytoplasmic tails of associatedJAK kinases being brought into proximity and facilitating thetrans-phosphorylation of tyrosine residues on the JAK kinase. Thistrans-phosphorylation results in the activation of the JAK kinase.

The downstream substrates of JAK family kinases include the signaltranducer activator of transcription (STAT) proteins. Phosphorylated JAKkinases bind various STAT (Signal Transducer and Activator ofTranscription) proteins. STAT proteins, which are DNA binding proteinsactivated by phosphorylation of tyrosine residues, function both assignaling molecules and transcription factors and ultimately bind tospecific DNA sequences present in the promoters of cytokine-responsivegenes (Leonard et al., (2000), J. Allergy Clin. Immunol. 105:877-888).

JAK/STAT signaling has been implicated in the mediation of many abnormalimmune responses such as allergies, asthma, autoimmune diseases such astransplant (allograft) rejection, rheumatoid arthritis, amyotrophiclateral sclerosis and multiple sclerosis, as well as in solid andhematologic malignancies such as leukemia and lymphomas. For a review ofthe pharmaceutical intervention of the JAK/STAT pathway see Frank,(1999), Mol. Med. 5:432:456 and Seidel et al., (2000), Oncogene19:2645-2656.

JAK3 in particular has been implicated in a variety of biologicalprocesses. For example, the proliferation and survival of murine mastcells induced by IL-4 and IL-9 have been shown to be dependent on JAK3-and gamma chain-signaling (Suzuki et al., (2000), Blood 96:2172-2180).JAK3 also plays a crucial role in IgE receptor-mediated mast celldegranulation responses (Malaviya et al., (1999), Biochem. Biophys. Res.Commun. 257:807-813), and inhibition of JAK3 kinase has been shown toprevent type I hypersensitivity reactions, including anaphylaxis(Malaviya et al., (1999), J. Biol. Chem. 274:27028-27038). JAK3inhibition has also been shown to result in immune suppression forallograft rejection (Kirken, (2001), Transpl. Proc. 33:3268-3270). JAK3kinases have also been implicated in the mechanism involved in early andlate stages of rheumatoid arthritis (Muller-Ladner et al., (2000), J.Immunal. 164:3894-3901); familial amyotrophic lateral sclerosis (Trieuet al., (2000), Biochem Biophys. Res. Commun. 267:22-25); leukemia(Sudbeck et al., (1999), Clin. Cancer Res. 5:1569-1582); mycosisfungoides, a form of T-cell lymphoma (Nielsen et al., (1997), Prac.Natl. Acad. Sci. USA 94:6764-6769); and abnormal cell growth (Yu et al.,(1997), J. Immunol. 159:5206-5210; Catlett-Falcone et al., (1999),Immunity 10: 105-115).

JAK1, JAK2, and TYK2 are expressed ubiquitously, whereas JAK3 isexpressed predominantly in hematopoietic cells. The JAK kinases,including JAK3, are abundantly expressed in primary leukemic cells fromchildren with acute lymphoblastic leukemia, the most common form ofchildhood cancer, and studies have correlated STAT activation in certaincells with signals regulating apoptosis (Demoulin et al., (1996), Mol.Cell. Biol. 16:4710-6; Jurlander et al., (1997), Blood. 89:4146-52;Kaneko et al., (1997), Clin. Exp. Immun. 109:185-193; and Nakamura etal., (1996), J. Biol. Chem. 271: 19483-8). They are also known to beimportant for lymphocyte differentiation, function and survival. JAK-3in particular plays an essential role in the function of lymphocytes,macrophages, and mast cells. Given the importance of this JAK kinase,compounds which modulate the JAK pathway, including those selective forJAK3, can be useful for treating diseases or conditions where thefunction of lymphocytes, macrophages, or mast cells is involved (Kudlaczet al., (2004) Am. J. Transplant 4:51-57; Changelian (2003) Science302:875-878). Conditions in which targeting of the JAK pathway ormodulation of the JAK kinases, particularly JAK3, are contemplated to betherapeutically useful include, leukemia, lymphoma, transplant rejection(e.g., pancreas islet transplant rejection, bone marrow transplantapplications (e.g., graft-versus-host disease), autoimmune diseases(e.g., diabetes, rheumatoid arthritis, lupus, psoriasis), andinflammation (e.g., asthma, allergic reactions). Conditions which canbenefit from JAK3 inhibition are discussed in greater detail below.Recent data on JAK inhibition has been reported in kidney allograftpatients treated with CP-690,550 and showed that markers of allogeneicresponse (interferon gamma) can be reduced (Van Gurp E A et al (2009)Transplanatation 87:79-86).

In view of the numerous conditions that are contemplated to benefit bytreatment involving modulation of the JAK pathway it is immediatelyapparent that new compounds that modulate JAK pathways and methods ofusing these compounds should provide substantial therapeutic benefits toa wide variety of patients. Provided herein are novel2,4-pyrimidinediamine compounds for use in the treatment of conditionsin which targeting of the JAK pathway or inhibition of JAK kinases,particularly JAK3, are therapeutically useful.

Patents and patent applications related to modulation of the JAK pathwayinclude: U.S. Pat. Nos. 5,728,536; 6,080,747; 6,080,748; 6,133,305;6,177,433; 6,210,654; 6,313,130; 6,316,635; 6,433,018; 6,486,185;6,506,763; 6,528,509; 6,593,357; 6,608,048; 6,610,688; 6,635,651;6,677,368; 6,683,082; 6,696,448; 6,699,865; 6,777,417; 6,784,195;6,825,190; 6,506,763; 6,784,195; 6,528,509; 6,608,048; 7,105,529;6,699,865; 6,825,190; 6,815,439; 6,949,580; 7,056,944; 6,998,391;7,074,793; 6,969,760; U.S. Pat. App. Pub. No. 2001/0007033 A1;2002/0115173 A1; 2002/0137141 A1; 2003/0236244 A1; 2004/0102455 A1;2004/0142404 A1; 2004/0147507 A1; and 2004/0214817 A1; and Internationalpatent applications WO 95/03701A1; WO 99/15500A1; WO 00/00202A1; WO00/10981A1; WO 00/47583A1; WO 00/51587A2; WO 00/55159A2; WO 01/42246A2;WO 01/45641A2; WO 01/52892A2; WO 01/56993A2; WO 01/57022A2; WO01/72758A1; WO 02/00661A1; WO 02/43735A1; WO 02/48336A2; WO 02/060492A1;WO 02/060927A1; WO 02/096909A1; WO 02/102800A1; WO 03/020698A2; WO03/048162A1; WO 03/101989A1; WO 2004/016597A2; WO 2004/041789A1; WO2004/041810A1; WO 2004/041814A1; WO 2004/046112A2; WO 2004/046120A2; WO2004/047843A1; WO 2004/058749A1; WO 2004/058753A1; WO 2004/085388A2; WO2004/092154A1; WO 2005/009957A1; WO 2005/016344A1; WO 2005/028475A2; andWO 2005/033107A1.

Patents and patent applications describing substituted pyrimidinediaminecompounds include: U.S. application Ser. No. 10/355,543 filed Jan. 31,2003 (US2004/0029902A 1), international application Serial No.PCT/US03/03022 filed Jan. 31, 2003 (WO 03/063794), U.S. application Ser.No. 10/631,029 filed Jul. 29, 2003, international application Serial No.PCT/US03/24087 (WO 04/014382), U.S. application Ser. No. 10/903,263filed Jul. 30, 2004, and international application Serial No.PCT/US2004/24716 (WO 05/016893), the disclosures of which areincorporated herein by reference. Substituted pyrimidinediaminecompounds are also described in international patent applicationpublication numbers: WO 02/059110, WO 03/074515, WO 03/106416, WO03/066601, WO 03/063794, WO 04/046118, WO 05/016894, WO 05/122294, WO05/066156, WO 03/002542, WO 03/030909, WO 00/39101, WO 05/037800 andU.S. Pat. Pub. No. 2003/0149064.

While progress has been made in this field, there remains a need in theart for compounds that inhibit syk and/or JAK kinase, as well as formethods for treating conditions in a patient, such as restenosis,thrombosis, and/or inflammation that can benefit from such inhibition.Moreover, the availability of compounds that selectively inhibit one ofthese kinases as compared to other kinases would also be desirable. Thepresent invention satisfies this and other needs.

BRIEF SUMMARY OF THE INVENTION

The present invention provides novel compounds having activity asinhibitors of syk activity (also referred to herein as “syk inhibitors”)and/or JAK kinase activity (also referred to herein as “JAKinhibitors”), as well as to methods for their preparation and use, andto pharmaceutical compositions containing the same. Such compounds havethe following structure (I):

or a pharmaceutically acceptable tautomer, salt, or stereoisomerthereof, wherein D¹, R¹, Y¹ and R² are as defined below.

The present invention also provides a pharmaceutical compositioncomprising a therapeutically effective amount of a compound of formulaI, or a pharmaceutical acceptable salt thereof, and a pharmaceuticallyacceptable carrier and/or diluent.

The compounds of the present invention have utility over a wide range oftherapeutic applications, and may be used to treat a variety ofconditions, mediated at least in part by syk activity, in both men andwomen, as well as a mammal in general (also referred to herein as a“subject”). For example, such conditions include, but are not limitedto, those associated with cardiovascular disease, inflammatory diseaseor autoimmune disease. More specifically, the compounds of the presentinvention have utility for treating conditions or disorders including,but not limited to: restenosis, thrombosis, inflammation, heparininduced thrombocytopenia, dilated cardiomyopathy, sickle cell disease,atherosclerosis, myocardial infarction, vascular inflammation, unstableangina, acute coronary syndromes, allergy, asthma, rheumatoid arthritis,B-cell mediated diseases such as Non Hodgkin's lymphoma,anti-phospholipid syndrome, lupus, psoriasis, multiple sclerosis, endstage renal disease, hemolytic anemia, immune thrombocytopenic purpura,and chronic lymphocytic leukemia. Thus, in one embodiment, methods aredisclosed which include the administration of an effective amount of acompound of formula (I), typically in the form of a pharmaceuticalcomposition, to a subject in need thereof.

The conditions associated with cardiovascular disease is selected fromthe group consisting of acute coronary syndrome, myocardial infarction,unstable angina, refractory angina, occlusive coronary thrombosisoccurring post-thrombolytic therapy or post-coronary angioplasty, athrombotically mediated cerebrovascular syndrome, embolic stroke,thrombotic stroke, transient ischemic attacks, venous thrombosis, deepvenous thrombosis, pulmonary embolism, coagulopathy, disseminatedintravascular coagulation, thrombotic thrombocytopenic purpura,thromboangiitis obliterans, thrombotic disease associated withheparin-induced thrombocytopenia, thrombotic complications associatedwith extracorporeal circulation, thrombotic complications associatedwith instrumentation such as cardiac or other intravascularcatheterization, intra-aortic balloon pump, coronary stent or cardiacvalve, and conditions requiring the fitting of prosthetic devices.

The present invention also provides a method for inhibiting the sykactivity of a blood sample comprising contacting said sample with acompound of the present invention.

The present invention further provides compounds in purified forms, aswell as chemical intermediates.

These and other aspects, objects, features and advantages of theinvention will be apparent upon reference to the following detaileddescription and figures. To this end, various references are set forthherein which describe in more detail certain background information,procedures, compounds and/or compositions, and are each herebyincorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows how Syk serves as a key mediator of Fc receptor mediatedsignaling in cellular biology and multiple diseases.

FIG. 2 shows how gene targeting of Syk indicated that Syk serves as akey mediator in arterial platelet biology and a selective target fortreating arterial thrombosis.

FIG. 3 shows a general synthesis of compounds of the present invention.

FIG. 4 provides table 1 illustrating compounds of the present inventionand syk IC₅₀s.

FIG. 5 provides table 2 illustrating compounds of the present inventionand syk IC₅₀s.

FIG. 6 provides table 3 illustrating compounds of the present inventionand syk IC₅₀s.

FIGS. 7A and B provide tables 4A and 4B illustrating compounds of thepresent invention and syk IC₅₀s.

FIG. 8 shows a series of compounds that were indented to selectivelyinhibit Syk in purified kinase assays. FIG. 8A) Compounds from theSyk-specific series (P459-72 and P505-15) and multi-kinase series(example 100b) were screened at 300 nM against the Millipore purifiedkinase panel (270 kinases tested with 10 μM ATP) to determine potencyand selectivity for Syk. Data are represented as a heat-map, defined atthe bottom. FIG. 8B) Subset of the purified kinases that had >80%inhibition by any of the three compounds. P459-72 only inhibited Syk andMLK1. P505-15 at 50 nM (˜10× greater than its Syk IC50) only inhibitedSyk. Example 100b inhibited multiple kinases, including Syk, JAK2 andJAK3. The IC50 of Syk inhibition is reported for each compound on theleft of the heat map. Percent kinase inhibition is given in each panelwithin the heat-map.

FIG. 9 shows the selective inhibition of Syk in non-Hodgkin's Lymphomacell lines. B cells were stimulated with anti-BCR antibody in thepresence of the indicated concentrations of Syk specific inhibitorsP459-72 and P505-15 (FIG. 9A and FIG. 9B) or the dual Syk/JAK inhibitorexample 100b (FIG. 9C). Western blot analyses of whole cell lysates werethen performed to evaluate Syk kinase activity (pBLNK Y84 and totalBLNK; top two gels) and Src kinase activity (pSyk Y352 and total Syk;bottom two gels). Experiments were performed 2-3 times each, bar graphsrepresent mean±S.D. of pBLNK Y84. The calculated IC50s of Syk kinaseinhibition are presented above the graphs.

FIG. 10 provides a comparison of Syk-Specific and Dual Syk/JAKInhibition in NHL Cell Lines. B cells were stimulated with anti-BCR(FIG. 10A), or IL-4 (FIG. 10B) for 15 min in the presence of variousconcentrations of each inhibitor, as indicated. Cells were thenevaluated for inhibition of signaling pathways by phospho-flowcytometry. FIG. 10A) bar graphs (mean±S.D., n=3) representing Srcactivity (pSyk Y352 MFI) and Syk activity (pERK Y204 MFI) following BCRstimulation under the various treatment conditions. FIG. 10B) Bar graphsdepicting pSTAT-6 Y641 MFI (mean±S.D., n=3) following stimulation withIL-4 in the presence of various concentrations of each inhibitor, asindicated.

FIG. 11 shows how Syk-specific inhibitors disrupt proliferation andsurvival of and induces apoptosis in NHL cell lines. The Syk-dependent“BCR type” and Syk-independent “non-BCR type” NHL cell lines werepreviously described (Polo, Juszczynski et al. Proc Natl Acad Sci USA104(9): 3207-12 (2007). FIG. 11A) Cells were treated for 72 h with 1 and3 μM of the Syk-specific inhibitor P505-15. Apoptosis was determined byFACS analysis of active caspase 3; data is represented as histograms.FIG. 11B) Additional cell lines were tested for sensitivity to Sykspecific (P459-72 and P505-15) versus dual Syk/JAK (example 100b)inhibition. Bar graphs represent mean±S.D. (n=3) of the percent ofcaspase 3 positive cells following each condition.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the below terms have the following meanings unlessspecified otherwise:

1. Abbreviations and Definitions

The abbreviations used herein are conventional, unless otherwisedefined. The following abbreviations are used: AcOH=acetic acid,AIBN=azobisisobutyronitrile (also azobisisobutylonitrile), aq.=aqueous,Boc=t-butylcarboxy, Bz—benzyl,BOP=benzotriazol-1-yloxytris(dimethylamino)-phosphoniumhexafluorophosphate, BPO=benzoyl peroxide, nBuOH=n-butanol,CBr₄=tetrabromomethane, mCPBA=m-chloroperoxybenzoic acid, CH₂Cl₂ orDCM=dichloromethane, Cs₂CO₃=cesium carbonate, CuCl₂=copper chloride;DIBAL=diisobutylaluminum hydride, DIEA=Hunig's base or diisopropylethylamine, DME=dimethyl ether, DMF=dimethyl formamide, DMSO=dimethylsulfoxide, DPPA=diphenyl phosphoryl azide, Et₃N=triethylamine,EtOAc=ethyl acetate, g=gram, HATU=2-(1H7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate,H₂=hydrogen; H₂O=water; HBr=hydrogen bromide; HCl=hydrogen chloride,HIV=human immunodeficiency virus, HPLC=high pressure liquidchromatography, h=hour, IgE=immunoglobulin E, IC₅₀=The concentration ofan inhibitor that is required for 50% inhibition of an enzyme in vitro,EPA=isopropyl alcohol, kg=kilogram, KCN=potassium cyanide, KOH=potassiumhydroxide, K₂PO₄=potassium phosphate, LDA=lithium diisopropylamine,LiAlH₄=lithium aluminum hydride=LiOH: lithium hydroxide;MeCN=acetonitrile; MS=Mass Spec, m/z=mass to charge ratio, MHz=MegaHertz, MeOH=methanol, μM=micromolar, μL=microliter, mg=milligram,mm=millimeter, mM=millimolar, mmol=millimole, mL=milliliter,mOD/min=millioptical density units per minute, min=minute, M=molar,Na₂CO₃=sodium carbonate, ng=nanogram, NaHCO₃=sodium bicarbonate;NaNO₂=sodium nitrite; NaOH=sodium hydroxide; Na₂S₂O₃=sodium bisulfate;Na₂SO₄=sodium sulfate; NBS=N-bromosuccinamide; NH₄Cl=ammonium chloride;NH₄OAc=ammonium acetate; NaSMe=sodium methylthiolate,NBS=N-bromosuccinamide, n-BuLi=n-butyl lithium, nm=nanometer,nM=nanomolar, N=Normal, NMP=N-methylpyrrolidine, NMR=nuclear magneticresonance, Pd/C=palladium on carbon,Pd(PPh₃)₄=Tetrakis-(triphenyl-phosphine)-palladium, pM=picomolar,Pin=pinacolato, PEG=polyethylene glycol, PPh₃ or Ph₃P=triphenylphosphine, RLV=Raucher leukemia virus, Ra—Ni=Rainey Nickel,SOCl₂=thionyl chloride, RT=room temperature, TEA=triethylamine,THF=tetrahydrofuran, TFA=trifluoroacetic acid, TLC=thin layerchromatography, TMS=trimethylsilyl, Tf=trifluoromethylsulfonyl andTSC=trisodium citrate.

It is noted here that as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise.

“Alkyl,” by itself or as part of another substituent, means, unlessotherwise stated, a straight or branched chain, fully saturatedaliphatic hydrocarbon radical having the number of carbon atomsdesignated. For example, “C₁₋₈alkyl” refers to a hydrocarbon radicalstraight or branched, containing from 1 to 8 carbon atoms that isderived by the removal of one hydrogen atom from a single carbon atom ofa parent alkane. The phrase “unsubstituted alkyl” refers to alkyl groupsthat do not contain groups other than fully saturated aliphatichydrocarbon radicals. Thus the phrase includes straight chain alkylgroups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl,octyl, nonyl, decyl, undecyl, dodecyl and the like. The phrase alsoincludes branched chain isomers of straight chain alkyl groups such asisopropyl, t-butyl, isobutyl, sec-butyl, and the like. Representativealkyl groups include straight and branched chain alkyl groups having 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. Furtherrepresentative alkyl groups include straight and branched chain alkylgroups having 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms.

“Alkenyl” by itself or as part of another substituent refers to astraight or branched chain, which may be mono- or polyunsaturated,having the number of carbon atoms designated. For example, “C₂-C₈alkenyl” means an alkenyl radical having from 2, 3, 4, 5, 6, 7 or 8atoms that is derived by the removal of one hydrogen atom from a singlecarbon atom of a parent alkane. Examples include, but are not limited tovinyl, 2-propenyl i.e. —CH═C(H)(CH₃), —CH═C(CH₃)₂, —C(CH₃)═C(H)₂,—C(CH₃)═C(H)(CH₃), —C(CH₂CH₃)═CH₂, butadienyl e.g. 2-(butadienyl),pentadienyl e.g. 2,4-pentadienyl and 3-(1,4-pentadienyl), andhexadienyl, among others, and higher homologs and stereoisomers thereof.A “substituted” alkenyl group includes alkenyl groups in which anon-carbon or non-hydrogen atom is bonded to a carbon double bonded toanother carbon and those in which one of the non-carbon or non-hydrogenatoms is bonded to a carbon not involved in a double bond to anothercarbon. Each site of unsaturation may be either cis or transconfiguration about the double bond(s).

The term “alkynyl”, by itself or as part of another substituent, means astraight or branched chain hydrocarbon radical, which may be mono- orpolyunsaturated, having the number of carbon atoms designated. Forexample, “C₂-C₈ alkynyl” means an alkynyl radical having from 2 to 8carbon atoms that is derived by the removal of one hydrogen atom from asingle carbon atom of a parent alkane. “Unsubstituted alkynyl” refers tostraight and branched chain groups such as those described with respectto unsubstituted alkyl groups as defined above, except that at least onetriple bond exists between two carbon atoms. Examples include, but arenot limited to ethynyl e.g. —C≡C(H), 1-propynyl e.g. —C≡C(CH₃),—C≡C(CH₂CH₃), —C(H₂)C≡C(H), —C(H)₂C≡C(CH₃), and —C(H)₂C≡C(CH₂CH₃) amongothers, and higher homologs and isomers thereof. A “substituted” alkynylgroup includes alkynyl groups in which a non-carbon or non-hydrogen atomis bonded to a carbon triple bonded to another carbon and those in whicha non-carbon or non-hydrogen atom is bonded to a carbon not involved ina triple bond to another carbon.

“Alkylene” by itself or as part of another substituent means a divalentradical derived from an alkane, as exemplified by —CH₂CH₂CH₂CH₂—.Typically, an alkylene group will have from 1, 2, 3, 4, 5, 6, 7 or 8carbon atoms that is derived by the removal of one hydrogen atom from asingle carbon atom of a parent alkyl.

“Cycloalkyl” or “carbocycle”, by themselves or in combination with otherterms, represent, unless otherwise stated, cyclic versions of “alkyl”,“alkenyl” and “alkynyl” in which all ring atoms are carbon. “Cycloalkyl”or “carbocycle” refers to a mono- or polycyclic group. When used inconnection with cycloalkyl substituents, the term “polycyclic” refersherein to fused and non-fused alkyl cyclic structures. “Cycloalkyl” or“carbocycle” may form a bridged ring or a spiro ring. The term“cycloalkenyl” refers to a cycloalkyl group that has at least one siteof alkenyl unsaturation between the ring vertices. The term“cycloalkynyl” refers to a cycloalkyl group that has at least one siteof alkynyl unsaturation between the ring vertices. When “cycloalkyl” isused in combination with “alkyl”, as in C₃₋₈cycloalkylC₃₋₈alkylene-, thecycloalkyl portion is meant to have the stated number of carbon atoms(e.g., from three to eight carbon atoms), while the alkylene portion hasfrom one to eight carbon atoms. Typical cycloalkyl substituents havefrom 3 to 8 ring atoms. Examples of cycloalkyl include cyclopentyl,cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like.

“Aryl” by itself or as part of another substituent refers to apolyunsaturated, aromatic, hydrocarbon group containing from 6 to 14carbon atoms, which can be a single ring or multiple rings (up to threerings) which are fused together or linked covalently. Thus the phraseincludes, but is not limited to, groups such as phenyl, biphenyl,anthracenyl, naphthyl by way of example. Non-limiting examples ofunsubstituted aryl groups include phenyl, 1-naphthyl, 2-naphthyl and4-biphenyl. “Substituted aryl group” includes, for example, —CH₂OH (onecarbon atom and one heteroatom replacing a carbon atom) and —CH₂SH. Theterm “heteroalkylene” by itself or as part of another substituent meansa divalent radical derived from heteroalkyl, as exemplified by—CH₂—CH₂—S—CH₂CH₂₋— and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkylene and heteroalkylene linkinggroups, no orientation of the linking group is implied.

The terms “heterocycle”, “heterocyclyl” or “heterocyclic” refer to asaturated or unsaturated non-aromatic cyclic group containing at leastone heteroatom. As used herein, the term “heteroatom” is meant toinclude oxygen (O), nitrogen (N), sulfur (S) and silicon (Si). Eachheterocycle can be attached at any available ring carbon or heteroatom.Each heterocycle may have one or more rings. When multiple rings arepresent, they can be fused together or linked covalently. Eachheterocycle typically contains 1, 2, 3, 4 or 5, independently selectedheteroatoms. Preferably, these groups contain 1, 2, 3, 4, 5, 6, 7, 8, 9or 10 carbon atoms, 0, 1, 2, 3, 4 or 5 nitrogen atoms, 0, 1 or 2 sulfuratoms and 0, 1 or 2 oxygen atoms. More preferably, these groups contain1, 2 or 3 nitrogen atoms, 0-1 sulfur atoms and 0-1 oxygen atoms.Non-limiting examples of heterocycle groups include morpholin-3-one,piperazine-2-one, piperazin-1-oxide, pyridine-2-one, piperidine,morpholine, piperazine, isoxazoline, pyrazoline, imidazoline,pyrazol-5-one, pyrrolidine-2,5-dione, imidazolidine-2,4-dione,pyrrolidine, tetrahydroquinolinyl, decahydroquinolinyl,tetrahydrobenzooxazepinyl dihydrodibenzooxepin and the like.

“Heteroaryl” refers to a cyclic or polycyclic aromatic radical thatcontain from one to five heteroatoms selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom or through acarbon atom and can contain 5 to 10 carbon atoms. Non-limiting examplesof heteroaryl groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,1-pyrazolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl,2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl and4-pyrimidyl. If not specifically stated, substituents for each of theabove noted aryl and heteroaryl ring systems are selected from the groupof acceptable substituents described herein. “Substituted heteroaryl”refers to a unsubstituted heteroaryl group as defined above in which oneor more of the ring members is bonded to a non-hydrogen atom such asdescribed above with respect to substituted alkyl groups and substitutedaryl groups. Representative substituents include straight and branchedchain alkyl groups-CH₃, —C₂H₅, —CH₂OH, —OH, —OCH₃, —OC₂H₅, —OCF₃,—OC(═O)CH₃, —OC(═O)NH₂, —OC(═O)N(CH₃)₂, —CN, —NO₂, —C(═O)CH₃, —CO₂H,—CO₂CH₃, —CONH₂, —NH₂, —N(CH₃)₂, —NHSO₂CH₃, —NHCOCH₃, —NHC(═O)OCH₃,—NHSO₂CH₃, —SO₂CH₃, —SO₂NH₂ and halo.

“Bicyclic heteroaryl” refers to bicyclic aromatic radical that containfrom one to five heteroatoms selected from N, O, and S, wherein thenitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A bicyclic heteroaryl group can beattached to the remainder of the molecule through a heteroatom orthrough a carbon atom and can contain 5 to 10 carbon atoms. Non-limitingexamples of bicyclic heteroaryl groups include 5-benzothiazolyl,purinyl, 2-benzimidazolyl, benzopyrazolyl, 5-indolyl, azaindole,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyland 6-quinolyl. If not specifically stated, substituents for each of theabove noted aryl and heteroaryl ring systems are selected from the groupof acceptable substituents described herein.

In each of the above embodiments designating a number of atoms e.g.“C₁₋₈” is meant to include all possible embodiments that have one feweratom. Non-limiting examples include C₁₋₇, C₂₋₈, C₂₋₇, C₃₋₈, C₃₋₇ and thelike.

Each of the terms herein (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) is meant to include both “unsubstituted” and optionally“substituted” forms of the indicated radical, unless otherwiseindicated. Typically each radical is substituted with 0, 1, 2 3 4 or 5substituents, unless otherwise indicated. Examples of substituents foreach type of radical are provided below.

“Substituted” refers to a group as defined herein in which one or morebonds to a carbon(s) or hydrogen(s) are replaced by a bond tonon-hydrogen and non-carbon atom “substituents” such as, but not limitedto, a halogen atom such as F, Cl, Br, and I; an oxygen atom in groupssuch as hydroxyl groups, alkoxy groups, aryloxy, and acyloxy groups; asulfur atom in groups such as thiol groups, alkyl and aryl sulfidegroups, sulfone groups, sulfonyl groups, and sulfoxide groups; anitrogen atom in groups such as amino, alkylamines, dialkylamines,arylamines, alkylarylamines, diarylamines, alkoxyamino, hydroxyamino,acylamino, sulfonylamino, N-oxides, imides, and enamines; and otherheteroatoms in various other groups. “Substituents” also include groupsin which one or more bonds to a carbon(s) or hydrogen(s) atom isreplaced by a higher-order bond (e.g., a double- or triple-bond) to aheteroatom such as oxygen in oxo, acyl, amido, alkoxycarbonyl,aminocarbonyl, carboxyl, and ester groups; nitrogen in groups such asimines, oximes, hydrazones, and nitriles. “Substituents” further includegroups in which one or more bonds to a carbon(s) or hydrogen(s) atoms isreplaced by a bond to a cycloalkyl, heterocyclyl, aryl, and heteroarylgroups. Representative “substituents” include, among others, groups inwhich one or more bonds to a carbon or hydrogen atom is/are replaced byone or more bonds to fluoro, chloro, or bromo group. Anotherrepresentative “substituent” is the trifluoromethyl group and othergroups that contain the trifluoromethyl group. Other representative“substituents” include those in which one or more bonds to a carbon orhydrogen atom is replaced by a bond to an oxygen atom such that thesubstituted alkyl group contains a hydroxyl, alkoxy, or aryloxy group.Other representative “substituents” include alkyl groups that have anamine, or a substituted or unsubstituted alkylamine, dialkylamine,arylamine, (alkyl)(aryl)amine, diarylamine, heterocyclylamine,diheterocyclylamine, (alkyl)(heterocyclyl)amine, or(aryl)(heterocyclyl)amine group. Still other representative“substituents” include those in which one or more bonds to a carbon(s)or hydrogen(s) atoms is replaced by a bond to an alkyl, cycloalkyl,aryl, heteroaryl, or heterocyclyl group.

The herein-defined groups may include prefixes and/or suffixes that arecommonly used in the art to create additional well-recognizedsubstituent groups. As examples, “alkylamino” refers to a group of theformula —NR^(a)R^(b). Unless stated otherwise, for the following groupscontaining R^(a), R^(b), R^(c), R^(d) and R^(e): R^(a), and R^(b) areeach independently selected from H, alkyl, alkoxy, thioalkoxy,cycloalkyl, aryl, heteroaryl, or heterocyclyl or are optionally joinedtogether with the atom(s) to which they are attached to form a cyclicgroup. When R^(a) and R^(b) are attached to the same nitrogen atom, theycan be combined with the nitrogen atom to form a 5-, 6- or 7-memberedring. For example, —NR^(a)R^(b) is meant to include 1-pyrrolidinyl and4-morpholinyl.

R^(c), R^(d), R^(e) and R^(f) are each independently selected fromalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl,heterocyclyl or alkylenearyl as defined herein.

Typically, a particular radical will have 0, 1, 2 or 3 substituents,with those groups having two or fewer substituents being preferred inthe present invention. More preferably, a radical will be unsubstitutedor monosubstituted. Most preferably, a radical will be unsubstituted.

“Substituents” for the alkyl and heteroalkyl radicals (as well as thosegroups referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl,alkynyl, cycloalkyl, heterocyclyl) can be a variety of groups selectedfrom: —OR^(a), ═O, ═NR^(a), ═N—OR^(a), —NR^(a)R^(b), —SR^(a), halogen,—SiR^(a)R^(b)R^(c), —OC(O)R^(a), —C(O)R^(a), —CO₂R^(a), —CONR^(a)R^(b),—OC(O)NR^(a)R^(b), —NR^(b)C(O)R^(a), —NR^(a)—C(O)NR^(b)R^(c),—NR^(a)—SO₂NR^(b)R^(c), —NR^(b)CO₂R^(a), —NH—C(NH₂)═NH,—NR^(a)C(NH₂)═NH, —NH—C(NH₂)═NR^(a), —S(O)R^(a), —SO₂R^(a),—SO₂NR^(a)R^(b), —NR^(b)SO₂R, —CN and —NO₂, in a number ranging fromzero to three, with those groups having zero, one or two substituentsbeing particularly preferred.

In some embodiments, “substituents” for the alkyl and heteroalkylradicals are selected from: —OR^(a), ═O, —NR^(a)R^(b), —SR^(a), halogen,—SiR^(a)R^(b)R^(c), —OC(O)R^(a), —C(O)R^(a), CO₂R^(a), —CONR^(a)R^(b),—OC(O)NR^(a)R^(b), —NR^(b)C(O)R^(a), —NR^(b)CO₂R^(a),—NR^(a)—SO₂NR^(b)R^(c), —S(O)R^(a), —SO₂R^(a), —SO₂NR^(a)R^(b),NR^(c)SO₂R, —CN and —NO₂, R^(a) are as defined above. In someembodiments, substituents are selected from: —OR^(a), ═O, —NR^(a)R^(b),halogen, —OC(O)R^(a), —CO₂R^(a), —CONR^(a)R^(b), —OC(O)NR^(a)R^(b), —NRBC(O)R^(a), —NR^(b)CO₂R^(a), —NR^(a)—SO₂NR^(b)R^(c), —SO₂R^(a),—SO₂NR^(a)R^(b), —NR″SO₂R, —CN and —NO₂.

Examples of substituted alkyl are: —(CH₂)₃NH₂, —(CH₂)₃NH(CH₃),—(CH₂)₃NH(CH₃)₂, —CH₂C(═CH₂)CH₂NH₂, —CH₂C(═O)CH₂NH₂, —CH₂S(═O)₂CH₃,—CH₂OCH₂NH₂, —CO₂H. Examples of substituents of substituted alkyl are:CH₂OH, —OH, —OCH₃, —OC₂H₅, —OCF₃, —OC(═O)CH₃, —OC(═O)NH₂,—OC(═O)N(CH₃)₂, —CN, —NO₂, —C(═O)CH₃, —CO₂H, —CO₂CH₃, —CONH₂, —NH₂,—N(CH₃)₂, —NHSO₂CH₃, —NHCOCH₃, —NHC(═O)OCH₃, —NHSO₂CH₃, —SO₂CH₃,—SO₂NH₂, and halo.

Similarly, “substituents” for the aryl and heteroaryl groups are variedand are selected from: -halogen, —OR^(a), —OC(O)R^(a), —NR^(a)R^(b),—SR^(a), —R^(a), —CN, —NO₂, —CO₂R^(a), —CONR^(a)R^(b), —C(O)R^(a),—OC(O)NR^(a)R^(b), —NR^(b)C(O)R^(a), —NR^(b)C(O)₂R^(a),—NR^(a)—C(O)NR^(b)R^(c), —NH—C(NH₂)═NH, —NR^(a)C(NH₂)═NH,—NH—C(NH₂)═NR^(a), —S(O)R^(a), —S(O)₂ R^(a), —S(O)₂NR^(a)R^(b), —N₃,—CH(Ph)₂, perfluoroC₁₋₈alkoxy, and perfluoroC₁₋₈alkyl, in a numberranging from zero to the total number of open valences on the aromaticring system; and where R^(a), R^(b) and R^(c) are independently selectedfrom hydrogen, C₁₋₆alkyl and heteroalkyl, unsubstituted aryl andheteroaryl, (unsubstituted aryl)-C₁₋₈alkyl, and (unsubstitutedaryl)oxy-C₁₋₈alkyl.

Two of the “substituents” on adjacent atoms of the aryl or heteroarylring may optionally be replaced with a substituent of the formula-T-C(O)—(CH₂)_(q)—U—, wherein T and U are independently —NH—, —O—, —CH₂—or a single bond, and q is 0, 1 or 2. Alternatively, two of thesubstituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula-A-(CH₂)_(r)—B—, wherein A and B are independently —CH₂—, —O—, —NH—,—S—, —S(O)—, —S(O)₂—, —S(O)₂NR^(a)— or a single bond, and r is 1, 2 or3. One of the single bonds of the new ring so formed may optionally bereplaced with a double bond. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula —(CH₂)_(S)—X—(CH₂)_(t)—, where s and tare independently integers of from 0 to 3, and X is —O—, —NR^(a)—, S—,—S(O)—, —S(O)₂, or —S(O)₂NR^(a)—. The substituent R^(a) in —NR^(a)— and—S(O)₂NR^(a)— is selected from hydrogen or unsubstituted C₁₋₁₆alkyl.Otherwise, R′ is as defined above.

Unless indicated otherwise, the nomenclature of substituents that arenot explicitly defined herein are arrived at by naming the terminalportion of the functionality followed by the adjacent functionalitytoward the point of attachment. For example, the substituent“arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O—C(O)—.

The term “acyl” refers to the group —C(═O)R^(c) where R^(c) is alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl orheterocyclyl. Acyl includes the “acetyl” group —C(═O)CH₃.

“Acylamino-” refers to the group —NR^(a)C(═O)R^(c) where R^(c) is alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl orheterocyclyl.

“Acyloxy” refers to —OC(═O)—R^(c) where R^(c) is alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl or heterocyclyl.

“Alkoxy” refers to —OR^(d) wherein R^(d) is alkyl as defined herein.Representative examples of alkoxy groups include methoxy, ethoxy,t-butoxy, trifluoromethoxy, and the like.

“Alkoxyamino” refers to the group —NHOR where R^(d) is alkyl.

“Alkoxycarbonyl” refers to —C(═O)OR^(d) wherein R^(d) is alkyl.Representative alkoxycarbonyl groups include, for example, those shownbelow.

These alkoxycarbonyl groups can be further substituted as will beapparent to those having skill in the organic and medicinal chemistryarts in conjunction with the disclosure herein.

“Alkoxycarbonylamino” refers to —NR^(a)C(═O)OR^(d) wherein R^(d) isalkyl

“Alkoxysulfonylamino” refers to the group-NR^(a)S(═O)₂—OR^(d) whereR^(d) is alkyl.

“Alkylcarbonyl” refers to the group —C(═O)R^(c) where R^(c) is alkyl.

“Alkylcarbonyloxy” refers to —OC(═O)—R^(c) where R^(c) is alkyl.

“Alkylcarbonylamino” refers to —NR^(a)C(═O)R^(c) wherein R^(c) is alkyl.Representative alkylcarbonylamino groups include, for example,—NHC(═O)CH₃, —NHC(═O)CH₂CH₃, —NHC(═O)CH₂NH(CH₃), —NHC(═O)CH₂N(CH₃)₂, or—NHC(═O)(CH₂)₃OH.

“Alkylsulfanyl”, “alkylthio”, or “thioalkoxy” refers to the groupS—R^(d). where R^(d) is alkyl.

“Alkylsulfonyl” refers to —S(═O)₂R^(e) where R^(e) is alkyl.Alkylsulfonyl groups employed in compounds of the present invention aretypically C₁₋₆alkylsulfonyl groups.

“Alkylsulfonylamino” refers to —NR^(a)S(═O)₂—R^(e) wherein R^(e) isalkyl.

“Alkynyloxy” refers to the group —O-alkynyl, wherein alkynyl is asdefined herein. Alkynyloxy includes, by way of example, ethynyloxy,propynyloxy, and the like.

“Amidino” refers to the group —C(═NR^(a))NR^(b)R^(c), wherein R^(b) andR^(c) independently are selected from the group consisting of hydrogen,alkyl, substituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl,cycloalkenyl, heteroaryl, heterocyclic, and where R^(b) and R^(c) areoptionally joined together with the nitrogen bound thereto to form aheterocyclic or substituted heterocyclic group. R^(a) is selected fromthe group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkynyl,aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic, substitutedheterocyclic, nitro, nitroso, hydroxy, alkoxy, cyano, —N═N—N-alkyl,—N(alkyl)SO₂-alkyl, —N═N═N-alkyl, acyl and —SO₂-alkyl.

“Amino” refers to a monovalent radical —NR^(a)R^(b) or divalent radical—NR^(a)—. The term “alkylamino” refers to the group —NR^(a)R^(b) whereR^(a) is alkyl and R^(b) is H or alkyl. The term “arylamino” refers tothe group —NR^(a)R^(b) where at least one R^(a) or R^(b) is aryl. Theterm “(alkyl)(aryl)amino” refers to the group —NR^(a)R^(b) where R^(a)is alkyl and R^(b) is aryl. Additionally, for dialkylamino groups, thealkyl portions can be the same or different and can also be combined toform a 3-7 membered ring with the nitrogen atom to which each isattached. Accordingly, a group represented as —NR^(a)R^(b) is meant toinclude piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl and the like.

“Aminocarbonyl” or “aminoacyl” refers to the amide —C(═O)—NR^(a)R^(b).The term “alkylaminocarbonyl” refers herein to the group—C(═O)—NR^(a)R^(b) where R^(a) is alkyl and R^(b) is H or alkyl. Theterm “arylaminocarbonyl” refers herein to the group —C(═O)—NR^(a)R^(b)where R^(a) or R^(b) is aryl. Representative aminocarbonyl groupsinclude, for example, those shown below. These aminocarbonyl group canbe further substituted as will be apparent to those having skill in theorganic and medicinal chemistry arts in conjunction with the disclosureherein.

“Aminocarbonylamino” refers to the group —NR^(a)C(O)NR^(a)R^(b), whereinR^(a) is hydrogen or alkyl and R^(a) and R^(b) independently areselected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl,aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic, and whereR^(a) and R^(b) are optionally joined together with the nitrogen boundthereto to form a heterocyclic or substituted heterocyclic group.

“Aminosulfonyl” refers to —S(O)₂NR^(a)R^(b) where R^(a) is independentlyare selected from the group consisting of hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, heteroaryl, substituted heteroaryl,heterocyclic, substituted heterocyclic and where R^(a) and R^(b) areoptionally joined together with the nitrogen bound thereto to form aheterocyclic or substituted heterocyclic group and alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic and substituted heterocyclic are as definedherein.

“Aminosulfonyloxy” refers to the group —O—SO₂NR^(a)R^(b), wherein R^(a)and R^(b) independently are selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl,heteroaryl and heterocyclic; R^(a) and R^(b) are optionally joinedtogether with the nitrogen bound thereto to form a heterocyclic orsubstituted heterocyclic group.

“Aminosulfonylamino” refers to the group —NR^(a)—SO₂NR^(b)R^(c), whereinR^(a) is hydrogen or alkyl and R^(b) and R^(c) independently areselected from the group consisting of hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, heteroaryl, substituted heteroaryl,heterocyclic, and substituted heterocyclic and where R^(b) and R^(c) areoptionally joined together with the nitrogen bound thereto to form aheterocyclic or substituted heterocyclic group, and wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic and substituted heterocyclic are as definedherein.

“Aminothiocarbonyl” refers to the group —C(S)NR^(a)R^(b), wherein R^(a)and R^(b) independently are selected from the group consisting ofhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,heteroaryl, substituted heteroaryl, heterocyclic, and substitutedheterocyclic and where R^(a) and R^(b) are optionally joined togetherwith the nitrogen bound thereto to form a heterocyclic or substitutedheterocyclic group, and wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Aminothiocarbonylamino” refers to the group —NR^(a)C(S)NR^(a)R^(b),wherein R^(a) is hydrogen or alkyl and R^(b) and R^(c) are optionallyjoined together with the nitrogen bound thereto to form a heterocyclicor substituted heterocyclic group.

“Arylcarbonyl” refers to the group —C(═O)R^(c) where R^(c) is aryl.

“Arylcarbonylamino” refers to —NR^(a)C(═O)R^(c) wherein R^(c) is aryl.

“Arylcarbonyloxy” refers to —OC(═O)—R^(c) where R^(c) is aryl.

“Aryloxy” refers to —OR^(d) where R^(d) is aryl. Representative examplesof aryloxy groups include phenoxy, naphthoxy, and the like.

“Aryloxycarbonyl” refers to —C(═O)OR^(d) wherein R^(d) is aryl.

“Aryloxycarbonylamino” refers to —NR^(a)C(═O)OR^(d) wherein R^(d) isaryl.

“Arylsulfanyl”, “arylthio”, or “thioaryloxy” refers to the groupS—R^(d). where R^(d) is aryl.

“Arylsulfonyl” refers to —S(═O)₂R^(e) where R^(e) is aryl.

“Arylsulfonylamino” refers to —NR^(a)S(═O)₂—R^(e) wherein R^(e) is aryl.

“Arylthio” refers to the group —S-aryl, wherein aryl is as definedherein. In other embodiments, sulfur may be oxidized to —S(O)— or —SO₂—moieties. The sulfoxide may exist as one or more stereoisomers.

“Azido” refers to —N₃.

“Bond” when used a element in a Markush group means that thecorresponding group does not exist, and the groups of both sides aredirectly linked.

“Carbonyl” refers to the divalent group —C(═O)—.

“Carboxy” or “carboxyl” refers to the group —CO₂H.

“Carboxyl ester” or “carboxy ester” refers to the groups —C(═O)OR^(c).

“(Carboxyl ester)amino” refers to the groups —NR^(a)—C(O)OR^(c), whereR^(a) is alkyl or hydrogen.

“(Carboxyl ester)oxy” or “Carbonate ester” refers to the groups—O—C(═O)OR^(c).

“Cyano” refers to —CN.

“Cycloalkoxy” refers to —OR^(d) where R^(d) is cycloalkyl.

“Cycloalkoxycarbonyl” refers to —C(═O)OR^(d) wherein R^(d) iscycloalkyl.

“Cycloalkoxycarbonylamino” refers to —NR^(a)C(═O)OR^(d) wherein R^(d) iscycloalkyl.

“Cycloalkylalkylene” refers to a radical —R^(x)R^(y) wherein R^(x) is analkylene group and R^(y) is a cycloalkyl group as defined herein, e.g.,cyclopropylmethyl, cyclohexenylpropyl, 3-cyclohexyl-2-methylpropyl, andthe like.

“Cycloalkylcarbonyl” refers to the group —C(═O)R^(c) where R^(c) iscycloalkyl.

“Cycloalkylcarbonylamino” refers to —NR^(a)C(═O)R^(c) wherein R^(c) iscycloalkyl.

“Cycloalkylcarbonyloxy” refers to —OC(═O)—R^(c) where R^(c) iscycloalkyl.

“Cycloalkylsulfonylamino” refers to —NR^(a)S(═O)₂—R^(e) wherein R^(e) iscycloalkyl.

“Cycloalkylthio” refers to —S-cycloalkyl. In other embodiments, sulfurmay be oxidized to —S(O)— or —SO₂— moieties. The sulfoxide may exist asone or more stereoisomers.

“Cycloalkenylox” refers to —O-cycloalkenyl.

“Cycloalkenylthio” refers to —S-cycloalkenyl. In other embodiments,sulfur may be oxidized to sulfinyl or sulfonyl moieties. The sulfoxidemay exist as one or more stereoisomers.

“Ester” refers to —C(═O)OR^(d) wherein R^(d) is alkyl, cycloalkyl, aryl,heteroaryl, or heterocyclyl.

“Guanidino” refers to the group —NHC(═NH)NH₂.

“Halo” or “halogen” by themselves or as part of another substituent,mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodineatom. Additionally, terms such as “haloalkyl”, are meant to includealkyl in which one or more hydrogen is substituted with halogen atomswhich can be the same or different, in a number ranging from one up tothe maximum number of halogens permitted e.g. for alkyl, (2m′+1), wherem′ is the total number of carbon atoms in the alkyl group. For example,the term “haloC₁₋₈alkyl” is meant to include trifluoromethyl,2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like. Theterm “perhaloalkyl” means, unless otherwise stated, alkyl substitutedwith (2m′+1) halogen atoms, where m′ is the total number of carbon atomsin the alkyl group. For example, the term “perhaloC₁₋₈alkyl”, is meantto include trifluoromethyl, pentachloroethyl,1,1,1-trifluoro-2-bromo-2-chloroethyl, and the like. Additionally, term“haloalkoxy” refers to an alkoxy radical substituted with one or morehalogen atoms.

“Heteroalkyl” means an alkyl radical as defined herein with one, two orthree substituents independently selected from cyano, —OR^(w),—NR^(x)R^(y), and —S(O)_(n)R^(z) (where n is an integer from 0 to 2),with the understanding that the point of attachment of the heteroalkylradical is through a carbon atom of the heteroalkyl radical. R^(w) ishydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl, aryl, aralkyl,alkoxycarbonyl, aryloxycarbonyl, carboxamido, or mono- ordi-alkylcarbamoyl. R^(x) is hydrogen, alkyl, cycloalkyl,cycloalkyl-alkyl, aryl or aralkyl. Ry is hydrogen, alkyl, cycloalkyl,cycloalkyl-alkyl, aryl, aralkyl, alkoxycarbonyl, aryloxycarbonyl,carboxamido, mono- or di-alkylcarbamoyl or alkylsulfonyl. R^(z) ishydrogen (provided that n is 0), alkyl, cycloalkyl, cycloalkyl-alkyl,aryl, aralkyl, amino, mono-alkylamino, di-alkylamino, or hydroxyalkyl.Representative examples include, for example, 2-hydroxyethyl,2,3-dihydroxypropyl, 2-methoxyethyl, benzyloxymethyl, 2-cyanoethyl, and2-methylsulfonyl-ethyl. For each of the above, R^(W), R^(x), R^(y), andR^(z) can be further substituted by amino, fluorine, alkylamino,di-alkylamino, OH or alkoxy. Additionally, the prefix indicating thenumber of carbon atoms (e.g., C₁-C₁₀) refers to the total number ofcarbon atoms in the portion of the heteroalkyl group exclusive of thecyano, —OR^(w), —NR^(x)R^(y), or —S(O)_(n)R^(z) portions.

“Heteroarylcarbonyl” refers to the group —C(═O)R^(c) where R^(c) isheteroaryl.

“Heteroarylcarbonylamino” refers to —NR^(a)C(═O)R^(c) wherein R^(c) isheteroaryl.

“Heteroarylcarbonyloxy” refers to —OC(═O)—R^(c) where R^(c) isheteroaryl.

“Heteroaryloxy” refers to —OR^(d) where R^(d) is heteroaryl.

“Heteroaryloxycarbonyl” refers to —C(═O)OR^(d) wherein R^(d) isheteroaryl.

“Heteroaryloxycarbonylamino” refers to —NR^(a)C(═O)OR^(d) wherein R^(d)is heteroaryl.

“Heteroarylsulfonyllamino” refers to —NR^(a)S(═O)₂—R^(e) wherein R^(e)is heteroaryl.

“Heteroarylthio” refers to the group —S-heteroaryl. In otherembodiments, sulfur may be oxidized to —S(O)— or —SO₂— moieties. Thesulfoxide may exist as one or more stereoisomers.

“Heterocyclylalkyl” or “Cycloheteroalkyl-alkyl” means a radical—R^(x)R^(y) where R^(x) is an alkylene group and R^(y) is a heterocyclylgroup as defined herein, e.g., tetrahydropyran-2-ylmethyl,4-(4-substituted-phenyl)piperazin-1-ylmethyl, 3-piperidinylethyl, andthe like.

“Heterocycloxycarbonylamino” refers to —NR^(a)C(═O)OR^(d) wherein R^(d)is heterocyclyl.

“Heterocyclylcarbonyl” refers to the —C(═O)R^(c) where R^(c) isheterocyclyl.

“Heterocyclylcarbonylamino” refers to —NR^(a)C(═O)R^(c) wherein R^(c) isheterocyclyl.

“Heterocyclylcarbonyloxy” refers to —OC(═O)—R^(c) where R^(c) sheterocyclyl.

“Heterocyclyloxy” refers to —OR^(d) where R^(d) is heterocyclyl.

“Heterocyclyloxycarbonyl” refers to —C(═O)OR^(d) wherein R^(d) isheterocyclyl.

“Heterocyclylsulfonyl” refers to —S(═O)₂R^(e) where R^(d) isheterocyclyl.

“Heterocyclylsulfonyllamino” refers to —NR^(a)S(═O)₂—R^(e) wherein R^(e)is heterocyclyl.

“Heterocyclylthio” refers to the group —S-heterocycyl. In otherembodiments, sulfur may be oxidized to —S(O)— or —SO₂— moieties. Thesulfoxide may exist as one or more stereoisomers.

“Hydroxy” or “hydroxyl” refers to the group —OH.

“Hydroxyamino” refers to the group —NHOH.

“Nitro” refers to —NO₂.

“Nitroso” refers to the group —NO.

The terms “optional” or “optionally” as used throughout thespecification means that the subsequently described event orcircumstance may but need not occur, and that the description includesinstances where the event or circumstance occurs and instances in whichit does not. For example, “heterocyclo group optionally mono- ordi-substituted with an alkyl group means that the alkyl may but need notbe present, and the description includes situations where theheterocyclo group is mono- or disubstituted with an alkyl group andsituations where the heterocyclo group is not substituted with the alkylgroup.

“Optionally substituted” means a ring which is optionally substitutedindependently with substituents. A site of a group that is unsubstitutedmay be substituted with hydrogen.

“Oxo” refers to the divalent group ═O.

“Sulfanyl” refers to the group —SR^(f) where R^(f) is as defined herein.

“Sulfinyl” refers to the group —S(═O)—R^(e) where R^(e) is as definedherein.

“Sulfonic acid” refers to the group —S(O)₂—OH.

“Sulfonyl” refers to the group —S(O)₂—R^(e) where R^(e) is as definedherein.

“Sulfonylamino” refers to —NR^(a)S(═O)₂—R^(e) where R^(a) is selectedfrom the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl,cycloalkyl, cycloalkenyl, heteroaryl and heterocyclyl and R^(e) is asdefined herein.

“Sulfonyloxy” refers to the group —OSO₂—R^(c).

Compounds that have the same molecular formula but differ in the natureor sequence of bonding of their atoms or the arrangement of their atomsin space are termed “isomers”. Isomers that differ in the arrangement oftheir atoms in space are termed “stereoisomers”. “Stereoisomer” and“stereoisomers” refer to compounds that exist in differentstereoisomeric forms if they possess one or more asymmetric centers or adouble bond with asymmetric substitution and, therefore, can be producedas individual stereoisomers or as mixtures. Stereoisomers includeenantiomers and diastereomers. Stereoisomers that are not mirror imagesof one another are termed “diastereomers” and those that arenon-superimposable mirror images of each other are termed “enantiomers”.When a compound has an asymmetric center, for example, it is bonded tofour different groups, a pair of enantiomers is possible. An enantiomercan be characterized by the absolute configuration of its asymmetriccenter and is described by the R- and S-sequencing rules of Cahn andPrelog, or by the manner in which the molecule rotates the plane ofpolarized light and designated as dextrorotatory or levorotatory (i.e.,as (+) or (−)-isomers respectively). A chiral compound can exist aseither individual enantiomer or as a mixture thereof. A mixturecontaining equal proportions of the enantiomers is called a “racemicmixture”. Unless otherwise indicated, the description is intended toinclude individual stereoisomers as well as mixtures. The methods forthe determination of stereochemistry and the separation of stereoisomersare well-known in the art (see discussion in Chapter 4 of ADVANCEDORGANIC CHEMISTRY, 4th edition J. March, John Wiley and Sons, New York,1992) differ in the chirality of one or more stereocenters.

“Thioacyl” refers to the groups R^(a)—C(S)—.

“Thiol” refers to the group —SH.

“Tautomer” refers to alternate forms of a molecule that differ in theposition of a proton, such as enol-keto and imine-enamine tautomers, orthe tautomeric forms of heteroaryl groups containing a —N═C(H)—NH— ringatom arrangement, such as pyrazoles, imidazoles, benzimidazoles,triazoles, and tetrazoles. A person of ordinary skill in the art wouldrecognize that other tautomeric ring atom arrangements are possible.

It is understood that in all substituted groups defined above, polymersarrived at by defining substituents with further substituents tothemselves (e.g., substituted aryl having a substituted aryl group as asubstituent which is itself substituted with a substituted aryl group,which is further substituted by a substituted aryl group, etc.) are notintended for inclusion herein. In such cases, the maximum number of suchsubstitutions is three. For example, serial substitutions of substitutedaryl groups are limited to -substituted aryl-(substitutedaryl)-substituted aryl.

“Protecting group” refers to a group of atoms that, when attached to areactive functional group in a molecule, mask, reduce or prevent thereactivity of the functional group. Typically, a protecting group may beselectively removed as desired during the course of a synthesis.Examples of protecting groups can be found in Greene and Wuts,Protective Groups in Organic Chemistry, 3^(rd) Ed., 1999, John Wiley &Sons, NY and Harrison et al., Compendium of Synthetic Organic Methods,Vols. 1-8, 1971-1996, John Wiley & Sons, NY. Representative aminoprotecting groups include, but are not limited to, formyl, acetyl,trifluoroacetyl, benzyl, benzyloxycarbonyl (“CBZ”), tert-butoxycarbonyl(“Boc”), trimethylsilyl (“TMS”), 2-trimethylsilyl-ethanesulfonyl(“TES”), trityl and substituted trityl groups, allyloxycarbonyl,9-fluorenylmethyloxycarbonyl (“FMOC”), nitro-veratryloxycarbonyl(“NVOC”) and the like. Representative hydroxy protecting groups include,but are not limited to, those where the hydroxy group is either acylatedor alkylated such as benzyl and trityl ethers, as well as alkyl ethers,tetrahydropyranyl ethers, trialkylsilyl ethers (e.g., TMS or TIPPSgroups) and allyl ethers.

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds which are prepared with relatively nontoxicacids or bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of salts derived frompharmaceutically-acceptable inorganic bases include aluminum, ammonium,calcium, copper, ferric, ferrous, lithium, magnesium, manganic,manganous, potassium, sodium, zinc and the like. Salts derived frompharmaceutically-acceptable organic bases include salts of primary,secondary and tertiary amines, including substituted amines, cyclicamines, naturally-occurring amines and the like, such as arginine,betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, malonic, benzoic, succinic, suberic,fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric,tartaric, methanesulfonic, and the like. Also included are salts ofamino acids such as arginate and the like, and salts of organic acidslike glucuronic or galactunoric acids and the like (see, e.g., Berge, S.M. et al., “Pharmaceutical Salts,” Journal of Pharmaceutical Science,66:1-19, 1977). Certain specific compounds of the present inventioncontain both basic and acidic functionalities that allow the compoundsto be converted into either base or acid addition salts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

In addition to salt forms, the present invention provides compoundswhich are in a prodrug ester form. “Prodrug”s of the compounds describedherein are those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent. Prodrugs arefrequently, but not necessarily, pharmacologically inactive untilconverted into the active drug. Prodrugs are typically obtained bymasking a functional group in the drug believed to be in part requiredfor activity with a progroup (defined below) to form a promoiety whichundergoes a transformation, such as cleavage, under the specifiedconditions of use to release the functional group, and hence the activedrug. The cleavage of the promoiety may proceed spontaneously, such asby way of a hydrolysis reaction, or it may be catalyzed or induced byanother agent, such as by an enzyme, by light, by acid or base, or by achange of or exposure to a physical or environmental parameter, such asa change of temperature. The agent may be endogenous to the conditionsof use, such as an enzyme present in the cells to which the prodrug isadministered or the acidic conditions of the stomach, or it may besupplied exogenously.

“Progroup” refers to a type of protecting group that, when used to maska functional group within an active drug to form a promoiety, convertsthe drug into a prodrug. Progroups are typically attached to thefunctional group of the drug via bonds that are cleavable underspecified conditions of use. Thus, a progroup is that portion of apromoiety that cleaves to release the functional group under thespecified conditions of use. As a specific example, an amide promoietyof the formula —NH—C(O)CH₃ comprises the progroup —C(O)CH₃.

A wide variety of progroups, as well as the resultant promoieties,suitable for masking functional groups in the active syk and/or JAKselective inhibitory compounds to yield prodrugs are well-known in theart. For example, a hydroxyl functional group may be masked as asulfonate, ester (such as acetate or maleate) or carbonate promoiety,which may be hydrolyzed in vivo to provide the hydroxyl group. An aminofunctional group may be masked as an amide, carbamate, imine, urea,phosphenyl, phosphoryl or sulfenyl promoiety, which may be hydrolyzed invivo to provide the amino group. A carboxyl group may be masked as anester (including methyl, ethyl, pivaloyloxymethyl, silyl esters andthioesters), amide or hydrazide promoiety, which may be hydrolyzed invivo to provide the carboxyl group. The invention includes those estersand acyl groups known in the art for modifying the solubility orhydrolysis characteristics for use as sustained-release or prodrugformulations. Other specific examples of suitable progroups and theirrespective promoieties will be apparent to those of skill in the art.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. “Solvate” refers toa complex formed by combination of solvent molecules with molecules orions of the solute. The solvent can be an organic compound, an inorganiccompound, or a mixture of both. Some examples of solvents include, butare not limited to, methanol, N,N-dimethylformamide, tetrahydrofuran,dimethylsulfoxide, and water. In general, the solvated forms areequivalent to unsolvated forms and are intended to be encompassed withinthe scope of the present invention. Certain compounds of the presentinvention may exist in multiple crystalline or amorphous forms. Ingeneral, all physical forms are equivalent for the uses contemplated bythe present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers, regioisomers and individual isomers (e.g., separateenantiomers) are all intended to be encompassed within the scope of thepresent invention. These isomers can be resolved or asymmetricallysynthesized using conventional methods to render the isomers “opticallypure”, i.e., substantially free of its other isomers. If, for instance,a particular enantiomer of a compound of the present invention isdesired, it may be prepared by asymmetric synthesis, or by derivationwith a chrial auxilliary, where the resulting diastereomeric mixture isseparated and the auxilliary group cleaved to provide the pure desiredenantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediasteromers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

The term “administering” refers to oral administration, administrationas a suppository, topical contact, intravenous, intraperitoneal,intramuscular, intralesional, intranasal or subcutaneous administration,or the implantation of a slow-release device e.g., a mini-osmotic pump,to a subject. Administration is by any route, including parenteral andtransmucosal (e.g., buccal, sublingual, palatal, gingival, nasal,vaginal, rectal, or transdermal). Parenteral administration includes,e.g., intravenous, intramuscular, intra-arteriole, intradermal,subcutaneous, intraperitoneal, intraventricular, and intracranial. Othermodes of delivery include, but are not limited to, the use of liposomalformulations, intravenous infusion, transdermal patches, etc.

An “agonist” or “activator” refers to an agent or molecule that binds toa receptor of the invention, stimulates, increases, opens, activates,facilitates, enhances activation or enzymatic activity, sensitizes or upregulates the activity of a receptor of the invention.

An “antagonist” or “inhibitor” refers to an agent or molecule thatinhibits or binds to, partially or totally blocks stimulation oractivity, decreases, closes, prevents, delays activation or enzymaticactivity, inactivates, desensitizes, or down regulates the activity of areceptor of the invention. As used herein, “antagonist” also includes areverse or inverse agonist.

As used herein, the term “condition or disorder responsive to modulationof syk and/or JAK” and related terms and phrases refer to a condition ordisorder associated with inappropriate, e.g., less than or greater thannormal, activity of syk and/or JAK and at least partially responsive toor affected by modulation of syk and/or JAK (e.g., syk and/or JAKantagonist or agonist results in some improvement in patient well-beingin at least some patients). Inappropriate functional activity of sykand/or JAK might arise as the result of expression of syk and/or JAK incells which normally do not express the receptor, greater than normalproduction of syk and/or JAK, or slower than normal metabolicinactivation or elimination of syk and/or JAK or its active metabolites,increased expression of syk and/or JAK or degree of intracellularactivation (leading to, e.g., inflammatory and immune-related disordersand conditions) or decreased expression of syk and/or JAK. A conditionor disorder associated with syk and/or JAK may include a “syk and/orJAK-mediated condition or disorder”.

As used herein, the phrases “a condition or disorder mediated at leastin part by syk or JAK kinase activity”, and related phrases and termsrefer to a condition or disorder characterized by inappropriate, e.g.,greater than normal, syk and/or JAK activity. Inappropriate syk and/orJAK functional activity might arise as the result of syk and/or JAKexpression in cells which normally do not express syk and/or JAK orincreased syk and/or JAK expression or degree of intracellularactivation (leading to, e.g., inflammatory and immune-related disordersand conditions). A condition or disorder mediated at least in part bysyk or JAK kinase activity may be completely or partially mediated byinappropriate syk and/or JAK functional activity. However, a conditionor disorder mediated at least in part by syk or JAK kinase activity isone in which modulation of syk and/or JAK results in some effect on theunderlying condition or disorder (e.g., an syk and/or JAK antagonistresults in some improvement in patient well-being in at least somepatients).

The term “inflammation” as used herein refers to infiltration of whiteblood cells (e.g., leukocytes, monocytes, etc.) into the area beingtreated for restenosis.

The term “intervention” refers to an action that produces an effect orthat is intended to alter the course of a disease process. For example,“vascular intervention” refers to the use of an intravascular proceduresuch as angioplasty or a stent to open an obstructed blood vessel.

The term “intravascular device” refers to a device useful for a vascularrecanalization procedure to restore blood flow through an obstructedblood vessel. Examples of intravascular devices include, withoutlimitation, stents, balloon catheters, autologous venous/arterialgrafts, prosthetic venous/arterial grafts, vascular catheters, andvascular shunts.

As used herein, the term “JAK” refers to a Janus kinase (RefSeqAccession No. P-43408) or a variant thereof that is capable of mediatinggene expression in vitro or in vivo. JAK variants include proteinssubstantially homologous to native JAK, i.e., proteins having one ormore naturally or non-naturally occurring amino acid deletions,insertions or substitutions (e.g., JAK derivatives, homologs andfragments). The amino acid sequence of JAK variant preferably is atleast about 80% identical to a native JAK, more preferably at leastabout 90% identical, and most preferably at least about 95% identical.

The term “leukocyte” refers to any of the various blood cells that havea nucleus and cytoplasm, separate into a thin white layer when wholeblood is centrifuged, and help protect the body from infection anddisease. Examples of leukocytes include, without limitation,neutrophils, eosinophils, basophils, lymphocytes, and monocytes.

The term “mammal” includes, without limitation, humans, domestic animals(e.g., dogs or cats), farm animals (cows, horses, or pigs), monkeys,rabbits, mice, and laboratory animals.

The terms “modulate”, “modulation” and the like refer to the ability ofa compound to increase or decrease the function and/or expression of sykand/or JAK, where such function may include transcription regulatoryactivity and/or protein-binding. Modulation may occur in vitro or invivo. Modulation, as described herein, includes the inhibition,antagonism, partial antagonism, activation, agonism or partial agonismof a function or characteristic associated with syk and/or JAK, eitherdirectly or indirectly, and/or the upregulation or downregulation of theexpression of syk and/or JAK, either directly or indirectly. In apreferred embodiment, the modulation is direct. Inhibitors orantagonists are compounds that, e.g., bind to, partially or totallyblock stimulation, decrease, prevent, inhibit, delay activation,inactivate, desensitize, or downregulate signal transduction. Activatorsor agonists are compounds that, e.g., bind to, stimulate, increase,open, activate, facilitate, enhance activation, activate, sensitize orupregulate signal transduction. The ability of a compound to inhibit thefunction of syk and/or JAK can be demonstrated in a biochemical assay,e.g., binding assay, or a cell-based assay, e.g., a transienttransfection assay.

“Modulators” of activity are used to refer to “ligands”, “antagonists”and “agonists” identified using in vitro and in vivo assays for activityand their homologs and mimetics. Modulators include naturally occurringand synthetic ligands, antagonists, agonists, molecules and the like.Assays to identify antagonists and agonists include, e.g., applyingputative modulator compounds to cells, in the presence or absence of areceptor of the invention and then determining the functional effects ona receptor of the invention activity. Samples or assays comprising areceptor of the invention that are treated with a potential activator,inhibitor, or modulator are compared to control samples without theinhibitor, activator, or modulator to examine the extent of effect.Control samples (untreated with modulators) are assigned a relativeactivity value of 100%. Inhibition is achieved when the activity valueof a receptor of the invention relative to the control is about 80%,optionally 50% or 25-1%. Activation is achieved when the activity valueof a receptor of the invention relative to the control is 110%,optionally 150%, optionally 200-500%, or 1000-3000% higher.

“Patient” refers to human and non-human animals, especially mammals.Examples of patients include, but are not limited to, humans, cows,dogs, cats, goats, sheep, pigs and rabbits.

Turning next to the compositions of the invention, the term“pharmaceutically acceptable carrier or excipient” means a carrier orexcipient that is useful in preparing a pharmaceutical composition thatis generally safe, non-toxic and neither biologically nor otherwiseundesirable, and includes a carrier or excipient that is acceptable forveterinary use as well as human pharmaceutical use. A “pharmaceuticallyacceptable carrier or excipient” as used in the specification and claimsincludes both one and more than one such carrier or excipient.

The terms “pharmaceutically effective amount”, “therapeuticallyeffective amount” or “therapeutically effective dose” refers to theamount of the subject compound that will elicit the biological ormedical response of a tissue, system, animal or human that is beingsought by the researcher, veterinarian, medical doctor or otherclinician. The term “therapeutically effective amount” includes thatamount of a compound that, when administered, is sufficient to preventdevelopment of, or alleviate to some extent, one or more of the symptomsof the condition or disorder being treated. The therapeuticallyeffective amount will vary depending on the compound, the disorder orcondition and its severity and the age, weight, etc., of the mammal tobe treated.

The term “platelet” refers to a minute, nonnucleated, disklike cellfound in the blood plasma of mammals that functions to promote bloodclotting.

The terms “prevent”, “preventing”, “prevention” and grammaticalvariations thereof as used herein, refers to a method of partially orcompletely delaying or precluding the onset or recurrence of a disorderor condition and/or one or more of its attendant symptoms or barring asubject from acquiring or reacquiring a disorder or condition orreducing a subject's risk of acquiring or reaquiring a disorder orcondition or one or more of its attendant symptoms.

The term “recanalization” refers to the process of restoring flow to orreuniting an interrupted channel of the body, such as a blood vessel.

The term “restenosis” refers to a re-narrowing or blockage of an arteryat the same site where treatment, such as an angioplasty or a stentprocedure, has been performed.

The phrase “selectively” or “specifically” when referring to binding toa receptor, refers to a binding reaction that is determinative of thepresence of the receptor, often in a heterogeneous population ofreceptors and other biologics. Thus, under designated conditions, thecompounds bind to a particular receptor at least two times thebackground and more typically more than 10 to 100 times background.Specific binding of a compound under such conditions requires a compoundthat is selected for its specificity for a particular receptor. Forexample, small organic molecules can be screened to obtain only thosecompounds that specifically or selectively bind to a selected receptorand not with other receptors or proteins. A variety of assay formats maybe used to select compounds that are selective for a particularreceptor. For example, High-throughput screening assays are routinelyused to select compounds that are selective for a particular a receptor.

As used herein, the term “Sickle cell anemia” refers to an inheriteddisorder of the red blood cells in which both hemoglobin alleles encodethe sickle hemoglobin (S) protein, i.e., the S/S genotype. The presenceof abnormal hemoglobin results in the production of unusually shapedcells, which do not survive the usual length of time in the bloodcirculation. Thus, anemia results. “Anemia” refers to a decrease in thenumber of red blood cells and/or hemoglobin in the blood.

The term “Sickle cell disease” refers to an inherited disorder of thered blood cells in which one hemoglobin allele encodes the sicklehemoglobin (S) protein, and the other allele encodes another unusualhemoglobin protein, such as hemoglobin (S), (C), (D), (E), and (βThal).Examples of sickle cell disease genotypes include, without limitation,the S/S, S/C, S/D, S/E, and S/βThal genotypes. The most common types ofsickle cell disease include sickle cell anemia, sickle-hemoglobin Cdisease, sickle beta-plus thalassemia, and sickle beta-zero thalassemia.

The “subject” is defined herein to include animals such as mammals,including, but not limited to, primates (e.g., humans), cows, sheep,goats, horses, dogs, cats, rabbits, rats, mice and the like. Inpreferred embodiments, the subject is a human.

As used herein, the term “syk” refers to a spleen tyrosine kinase(RefSeq Accession No. P-043405) or a variant thereof that is capable ofmediating a cellular response to T-cell receptors in vitro or in vivo.syk variants include proteins substantially homologous to native syk,i.e., proteins having one or more naturally or non-naturally occurringamino acid deletions, insertions or substitutions (e.g., sykderivatives, homologs and fragments). The amino acid sequence of sykvariant preferably is at least about 80% identical to a native syk, morepreferably at least about 90% identical, and most preferably at leastabout 95% identical.

The term “syk inhibitor” refers to any agent that inhibits the catalyticactivity of spleen tyrosine kinase.

The term “thrombosis” refers to the blockage or clotting of a bloodvessel caused by a clumping of cells, resulting in the obstruction ofblood flow. The term “thrombosis” refers to the clot that is formedwithin the blood vessel.

The terms “treat”, “treating”, “treatment” and grammatical variationsthereof as used herein, includes partially or completely delaying,alleviating, mitigating or reducing the intensity of one or moreattendant symptoms of a disorder or condition and/or alleviating,mitigating or impeding one or more causes of a disorder or condition.Treatments according to the invention may be applied preventively,prophylactically, pallatively or remedially.

The term “vessel” refers to any channel for carrying a fluid, such as anartery or vein. For example, a “blood vessel” refers to any of thevessels through which blood circulates in the body. The lumen of a bloodvessel refers to the inner open space or cavity of the blood vessel.

2. Embodiments of the Invention a. Compounds

The present invention provides in one embodiment, a compound having theformula I:

or a pharmaceutically acceptable salt thereof, wherein:

D¹ is selected from the group consisting of

(a) C₃₋₈cycloalkyl, optionally substituted with from 1 to 4 substituentsindependently selected from the group consisting of: C₁₋₈ alkyl, amino,hydroxy, C₁₋₈alkylcarbonyl, aminocarbonyl, C₁₋₈alkoxycarbonylamino,arylC₁₋₈alkoxycarbonylamino, phenyl and heterocyclylC₁₋₈alkylene;

(b) C₁₋₈ alkyl; optionally substituted with from 1 to 4 substituentsindependently selected from the group consisting of: amino, oxo,C₁₋₈alkoxy, C₂₋₈alkynyl, cyano, aminocarbonyl, C₁₋₈haloalkyl, hydroxy,halogen, C₃₋₈cycloalkyl, and phenyl;

(c) C₁₋₈ alkylC₃₋₈heterocyclyl; optionally substituted with from 1 to 4substituents independently selected from the group consisting of:C₁₋₈alkyl, C₁₋₈alkylcarbonyl; C₁₋₈alkylsulfonyl; aminocarbonyl

(d) aryl, which is optionally substituted with from 1 to 4 substituentsindependently selected from the group consisting of C₁₋₈alkyl,C₂₋₈alkenyl, C₂₋₈alkynyl, C₁₋₈haloalkyl, carboxy, acyl, acylamino,cyano, amino, aminocarbonyl, aminosulfonyl, sulfonyl, nitro, hydroxy,C₁₋₈alkoxy, aryloxy, halo, sulfonylamino, C₃₋₈cycloalkyl, aryl,heterocyclyl C₁₋₈alkylsulfonyl, C₁₋₈alkylcarbonylheterocyclyl andheteroaryl;

(e) heteroaryl, optionally substituted with from 1 to 4 substituentsindependently selected from the group consisting of: C₁₋₈alkyl,C₁₋₈alkylcarbonyl, aminocarbonyl, C₁₋₈alkoxycarbonyl, amino, C₁₋₈alkoxycarbonylamino, arylC₁₋₈alkoxycarbonylamino, hydroxyl, C₁₋₈ alkoxy,C₁₋₈alkylsulfonyl, oxo, halo, phenyl and heterocyclylC₁₋₈alkylene;

(f) C₃₋₈heterocyclyl, optionally substituted with from 1 to 4substituents independently selected from the group consisting of:C₁₋₈alkyl, C₁₋₈alkoxycarbonyl and oxo;

R¹ is selected from the group consisting of H, C₁₋₈ alkyl, amino,aminocarbonyl, hydroxyl, C₁₋₈ alkoxy, C₁₋₈ haloalkyl, C₂₋₈ alkenyl, C₂₋₈alkynyl, oxo, cyano, C₁₋₈ alkoxycarbonyl, C₃₋₈ cycloalkyl, aryl andheterocyclyl; and each heterocyclyl is optionally substituted with from1 to 4 substituents selected from the group consisting of: C₁₋₈ alkyl,halo, oxo, amino, C₁₋₈alkoxy, C₁₋₈alkylcarbonyl, arylC₁₋₈alkoxycarbonyl, aminocarbonyl, arylC₁₋₈ alkylenecarbonyl and C₁₋₈alkylsulfonyl

Y¹ is selected from the group consisting of

(a) aryl; optionally substituted with from 1 to 3 substituents, R^(4a),independently selected from the group consisting of C₁₋₈alkyl,C₁₋₈alkoxyC₁₋₈alkyl, aminocarbonyl-, hydroxyl, oxo, halogen, hydroxy,C₁₋₈alkoxy and C₁₋₈alkylsulfonyl;

(b) heteroaryl, optionally substituted with from 1 to 3 substituents,R^(4a), independently selected from the group consisting of C₁₋₈alkyl,C₁₋₈alkoxyC₁₋₈alkyl, aminocarbonyl-, hydroxyl, oxo, halogen, hydroxy,C₁₋₈alkoxy and C₁₋₈alkylsulfonyl;

R² is selected from the group consisting of heterocyclyl,heterocyclylaminosulfonyl, heterocyclylcarbonyl,heterocyclylcarbonylC₁₋₈alkoxy, heterocyclyl C₁₋₈alkoxy, heteroaryl,aminosulfonyl, C₁₋₈alkysulfinyl, C₁₋₈haloalkoxy,C₁₋₈alkoxycarbonylamino, C₁₋₈alkoxyaminocarbonyl,aminocarbonylC₁₋₈alkylamino, aminosulfonylheterocyclyl andalkylcarbonylheterocyclyl;

and wherein R² is further optionally substituted with from 1 to 2substituents, R^(2c), independently selected from the group consistingof C₁₋₈ alkyl, C₁₋₈alkoxy, halo, aminocarbonyl, oxo, hydroxyl,aminoC₁₋₈alkylene, C₁₋₈alkoxyC₁₋₈alkylene, C₁₋₈alkylcarbonyl,C₃₋₈cycloalkylcarbonyl, heterocyclylcarbonyl, C₁₋₈alkylcarbonylamino,C₃₋₈cycloalkylcarbonylamino, heterocyclylcarbonylamino,C₁₋₈alkylsulfonyl, C₃₋₈cycloalkylsulfonyl, heterocyclylsulfonyl,C₃₋₈cycloalkyl, C₁₋₈alkylcycloalkylene, heteroaryl.

In one group of embodiments, Y¹ is aryl. In another group ofembodiments, Y¹ is phenyl. In another group of embodiments, Y¹ isheteroaryl. In another group of embodiments, Y¹ is pyridinyl.

In one group of embodiments, Y¹ is aryl. In another group ofembodiments, Y¹ is phenyl. In another group of embodiments, Y¹ isheteroaryl. In another group of embodiments, Y¹ is pyridinyl.

In one group of embodiments, R² is heterocyclyl. In another group ofembodiments, R² is heterocyclylaminosulfonyl. In another group ofembodiments, R² is heterocyclylcarbonyl. In another group ofembodiments, R² is heterocyclylcarbonylC₁₋₈alkoxy. In another group ofembodiments, R² is heterocyclylC₁₋₈alkoxy. In another group ofembodiments, R² is heteroaryl. In another group of embodiments, R² isaminosulfonyl. In another group of embodiments, R² is C₁₋₈alkysulfinyl.In another group of embodiments, R² is C₁₋₈haloalkoxy. In another groupof embodiments, R² is C₁₋₈alkoxycarbonylamino. In another group ofembodiments, R² is C₁₋₈alkoxyaminocarbonyl. In another group ofembodiments, R² is aminocarbonylC₁₋₈alkylamino.

In one group of embodiments, if R² is a heterocyclyl or heteroaryl it issubstituted with at least one group, R³, selected from the groupconsisting of aminoC₁₋₈alkyl-, C₁₋₈alkoxyC₁₋₈alkyl-, oxo-,C₁₋₈alkylcarbonyl, C₃₋₈cycloalkylcarbonyl, heterocyclylcarbonyl,C₁₋₈alkylcarbonylamino, C₃₋₈cycloalkylcarbonylamino,heterocyclylcarbonylamino, C₁₋₈alkylsulfonyl, C₃₋₈cycloalkylsulfonyl andheterocyclylsulfonyl.

In another group of embodiments, D¹ is C₁₋₈ alkyl. In another group ofembodiments, D¹ is C₁₋₈ alkylC₃₋₈heterocyclyl.

In another group of embodiments, D¹ is aryl. In another group ofembodiments, D¹ is phenyl.

In another group of embodiments, D¹ is heteroaryl.

The present invention provides in another group of embodiments, acompound wherein a heteroaryl group of the compound of formula I isselected from the group consisting of:

each of which is optionally substituted with from 1 to 2 substituentsindependently selected from the group consisting of: C₁₋₈ alkyl, amino,hydroxyl, oxo, halo, C₁₋₈ alkoxy, hydroxyC₁₋₈alkyl-, aminoC₁₋₈alkyl,C₁₋₈alkylcarbonyl, haloC₁₋₈alkyl, C₃₋₈cycloalkyl, C₁₋₈aminocycloalkyl,aminoC₁₋₈alkylenecarbonyl, aminocarbonyl,C₁₋₈alkyleneaminoC₁₋₈alkylenecarbonyl, C₁₋₈alkoxyC₁₋₈alkylenecarbonyl,hydroxyC₁₋₈alkylenecarbonyl, hydroxyC₁₋₈alkoxycarbonyl, C₁₋₈alkoxycarbonylamino, aryl, arylC₁₋₈ alkoxycarbonylamino,C₁₋₈alkylsulfonyl, aminoC₁₋₈alkylenesulfonyl, aminosulfonyl,C₁₋₈alkyleneaminoC₁₋₈alkylenesulfonyl, C₁₋₈alkoxyC₁₋₈alkylenesulfonyl,hydroxyC₁₋₈alkylenesulfonyl, hydroxyC₁₋₈alkoxysulfonyl, aminosulfonyl,and C₁₋₈alkylheterocyclyl.

The present invention provides in another group of embodiments, acompound wherein a heteroaryl group of the compound of formula I is apolycyclic heteroaryl group selected from the group consisting of:

optionally substituted with from 1 to 3 substituents independentlyselected from the group consisting of: C₁₋₈ alkyl, C₁₋₈alkylcarbonyl,C₁₋₈aminocycloalkyl, aminoC₁₋₈alkylenecarbonyl, aminocarbonyl,C₁₋₈alkyleneaminoC₁₋₈alkylenecarbonyl, C₁₋₈alkoxyC₁₋₈alkylenecarbonyl,hydroxyC₁₋₈alkylenecarbonyl, hydroxyC₁₋₈alkoxycarbonyl, aminocarbonyl,amino, C₁₋₈ alkoxycarbonylamino, aryl, arylC₁₋₈ alkoxycarbonylamino,hydroxyl, C₁₋₈ alkoxy, C₁₋₈alkylsulfonyl, aminoC₁₋₈alkylenesulfonyl,aminosulfonyl, C₁₋₈alkyleneaminoC₁₋₈alkylenesulfonyl,C₁₋₈alkoxyC₁₋₈alkylenesulfonyl, hydroxyC₁₋₈alkylenesulfonyl,hydroxyC₁₋₈alkoxysulfonyl, aminosulfonyl, oxo, halo, phenyl andC₁₋₈alkylheterocyclyl; and the wavy line indicates the point ofattachment to the rest of the molecule.

The present invention provides in another group of embodiments, acompound wherein: D¹ is bicyclic heteroaryl. In another group ofembodiments, D¹ is selected from the group consisting of:

optionally substituted with from 1 to 3 substituents independentlyselected from the group consisting of: C₁₋₈ alkyl, C₁₋₈alkylcarbonyl,C₁₋₈aminocycloalkyl, aminoC₁₋₈alkylenecarbonyl, aminocarbonyl,C₁₋₈alkyleneaminoC₈alkylenecarbonyl, C₁₋₈alkoxyC₁₋₈alkylenecarbonyl,hydroxyC₁₋₈alkylenecarbonyl, hydroxyC₁₋₈alkoxycarbonyl, aminocarbonyl,amino, C₁₋₈ alkoxycarbonylamino, aryl, arylC₁₋₈ alkoxycarbonylamino,hydroxyl, C₁₋₈ alkoxy, C₁₋₈alkylsulfonyl, aminoC₁₋₈alkylenesulfonyl,aminosulfonyl, C₁₋₈alkyleneaminoC₁₋₈alkylenesulfonyl,C₁₋₈alkoxyC₁₋₈alkylenesulfonyl, hydroxyC₁₋₈alkylenesulfonyl,hydroxyC₁₋₈alkoxysulfonyl, aminosulfonyl, oxo, halo, phenyl andC₁₋₈alkylheterocyclyl; and the wavy line indicates the point ofattachment to the rest of the molecule.

The present invention provides in another group of embodiments, acompound wherein D¹ is C₃₋₈heterocyclyl. In one group of embodiments,any of the heterocyclyl groups of formula I is selected from the groupconsisting of:

In another group of embodiments, D¹ is selected from the groupconsisting of:

each of which is optionally substituted with from 1 to 3 substituentsindependently selected from the group consisting of: C₁₋₈alkyl,C₁₋₈alkoxycarbonyl and oxo; and the wavy line indicates the point ofattachment to the rest of the molecule.

The present invention provides in one embodiment, a compound having theformula I:

or a pharmaceutically acceptable tautomer, salt, or stereoisomerthereof, wherein:

D¹ is selected from the group consisting of

(a) C₁₋₈ alkyl; optionally substituted with from 1 to 4 substituentsindependently selected from the group consisting of: C₁₋₈alkoxy,C₂₋₈alkynyl, cyano, aminocarbonyl, C₁₋₈haloalkyl, hydroxy,C₃₋₈cycloalkyl, C₃₋₈heterocyclyl and phenyl;

(b) C₃₋₈cycloalkyl, optionally substituted with from 1 to 4 substituentsindependently selected from the group consisting of: C₁₋₈ alkyl, amino,hydroxy, C₁₋₈alkylcarbonyl, aminocarbonyl, C₁₋₈alkoxycarbonylamino,arylC₁₋₈alkoxycarbonylamino, phenyl and heterocyclylC₁₋₈alkylene;

(c) aryl, which is optionally substituted with from 1 to 4 substituentsindependently selected from the group consisting of C₁₋₈alkyl,C₂₋₈alkenyl, C₂₋₈alkynyl, C₁₋₈haloalkyl, carboxy, acyl, acylamino,cyano, amino, aminocarbonyl, aminosulfonyl, sulfonyl, nitro, hydroxy,C₁₋₈alkoxy, aryloxy, halo, sulfonylamino, C₃₋₈cycloalkyl, aryl,heterocyclyl C₁₋₈alkylsulfonyl, C₁₋₈alkylcarbonylheterocyclyl andheteroaryl;

(d) heteroaryl, optionally substituted with from 1 to 4 substituentsindependently selected from the group consisting of: C₁₋₈alkyl,C₁₋₈alkylcarbonyl, aminocarbonyl, C₁₋₈alkoxycarbonyl, amino, C₁₋₈alkoxycarbonylamino, arylC₁₋₈alkoxycarbonylamino, hydroxyl, C₁₋₈ alkoxy,C₁₋₈alkylsulfonyl, oxo, halo, phenyl and heterocyclylC₁₋₈alkylene;

(e) C₃₋₈heterocyclyl, optionally substituted with from 1 to 4substituents independently selected from the group consisting of:C₁₋₈alkyl, C₁₋₈alkoxycarbonyl and oxo;

R¹ is selected from the group consisting of H, C₁₋₈ alkyl, amino,aminocarbonyl, hydroxyl, C₁₋₈ alkoxy, C₁₋₈ haloalkyl, C₂₋₈ alkenyl, C₂₋₈alkynyl, oxo, cyano, C₁₋₈ alkoxycarbonyl, C₃₋₈ cycloalkyl, aryl andheterocyclyl; and each heterocyclyl is optionally substituted with from1 to 4 substituents selected from the group consisting of: C₁₋₈ alkyl,halo, oxo, amino, C₁₋₈alkoxy, C₁₋₈alkylcarbonyl, arylC₁₋₈alkoxycarbonyl, aminocarbonyl, arylC₁₋₈ alkylenecarbonyl and C₁₋₈alkylsulfonyl

Y¹ is selected from the group consisting of

(a) aryl; optionally substituted with from 1 to 3 substituents, R^(4a),independently selected from the group consisting of C₁₋₈alkyl,C₁₋₈alkoxyC₁₋₈alkyl, aminocarbonyl-, hydroxyl, oxo, halogen, hydroxy,C₁₋₈alkoxy and C₁₋₈alkylsulfonyl;

(b) heteroaryl, optionally substituted with from 1 to 3 substituents,R^(4a), independently selected from the group consisting of C₁₋₈alkyl,C₁₋₈alkoxyC₁₋₈alkyl, aminocarbonyl-, hydroxyl, oxo, halogen, hydroxy,C₁₋₈alkoxy and C₁₋₈alkylsulfonyl;

R² is selected from the group consisting of aminosulfonyl,C₁₋₈alkysulfinyl, C₁₋₈haloalkoxy, C₁₋₈alkoxycarbonylamino andheterocyclyl; wherein if R² is heterocyclyl it is substituted with atleast one group, R³, selected from the group consisting of aminC₁₋₈alkyl-, C₁₋₈alkoxyC₁₋₈alkyl-, oxo-, C₁₋₈alkylcarbonyl,C₃₋₈cycloalkylcarbonyl, heterocyclylcarbonyl, C₁₋₈alkylcarbonylamino,C₃₋₈cycloalkylcarbonylamino, heterocyclylcarbonylamino,C₁₋₈alkylsulfonyl, C₃₋₈cycloalkylsulfonyl, heterocyclylsulfonyl; andwherein R² is further optionally substituted with from 1 to 2substituents, R^(4c), independently selected from the group consistingof C₁₋₈ alkyl, C₁₋₈alkoxy, halo, aminocarbonyl, oxo, hydroxyl,aminoC₁₋₈alkylene, C₁₋₈alkoxyC₁₋₈alkylene, C₁₋₈alkylcarbonyl,C₃₋₈cycloalkylcarbonyl, heterocyclylcarbonyl, C₁₋₈alkylcarbonylamino,C₃₋₈cycloalkylcarbonylamino, heterocyclylcarbonylamino,C₁₋₈alkylsulfonyl, C₃₋₈cycloalkylsulfonyl, heterocyclylsulfonyl,C₃₋₈cycloalkyl and C₁₋₈alkylcycloalkylene.

The present invention provides in another embodiment, a compound whereinthe moiety —Y¹—R² is selected from the group consisting of:

Y 2 is N, CH or C;

R^(4a) is selected from the group consisting of H and C₁₋₈alkyl;

each R^(4c) is independently selected from the group consisting of H,C₁₋₈alkyl, C₁₋₈alkoxyC₁₋₈alkyl, aminocarbonyl-, hydroxyl, oxo,C₁₋₈alkoxy and halo;

each R^(4b) is independently selected from the group consisting of H,C₁₋₈alkyl, amino, C₁₋₈alkoxy and heterocyclyl;

X is halo;

the subscript p is 0, 1, 2 or 3; and

-   -   the wavy line indicates the point of attachment to the rest of        the molecule.

The present invention provides in another embodiment, a compound whereinthe moiety —Y¹—R² is selected from the group consisting of:

and the wavy line indicates the point of attachment to the rest of themolecule.

The present invention provides in another embodiment, a compound havingthe formula Ia:

-   -   or a pharmaceutically acceptable tautomer, salt, or stereoisomer        thereof.

The present invention provides in another embodiment, a compound havingthe formula Ib:

-   -   or a pharmaceutically acceptable tautomer, salt, or stereoisomer        thereof.

The present invention provides in another embodiment, a compound havingthe formula Ic:

-   -   or a pharmaceutically acceptable salt, stereoisomer or prodrug        thereof.

The present invention provides in another embodiment, a compound havingthe formula Id:

-   -   or a pharmaceutically acceptable salt, stereoisomer or prodrug        thereof.

The present invention provides in another embodiment, a compound havingthe formula Id1:

-   -   or a pharmaceutically acceptable salt, stereoisomer or prodrug        thereof.

The present invention provides in another embodiment, a compound havingthe formula Id2:

-   -   or a pharmaceutically acceptable salt, stereoisomer or prodrug        thereof.

The present invention provides in another embodiment, a compoundwherein,

D¹ is C₁₋₈ alkyl; optionally substituted with from 1 to 4 substituentsindependently selected from the group consisting of: C₁₋₈alkoxy,C₂₋₈alkynyl, CN, aminocarbonyl, C₁₋₈haloalkyl, -hydroxyl,—C₃₋₈cycloalkyl, —C₃₋₈heterocyclyl, and phenyl;

R¹ is selected from the group consisting of H, C₁₋₈ alkyl, amino,hydroxyl, C₁₋₈ alkoxy, C₁₋₈ haloalkyl, C₂₋₈ alkenyl, C₂₋₈ alkynyl, oxo,CN, C₁₋₈ alkoxycarbonyl, C₃₋₈ cycloalkyl, aryl, heterocyclyl; eachheterocyclyl is optionally substituted with from 1 to 4 substituentsselected from the group consisting of: alkyl, halo, oxo, amino, alkoxy,C₁₋₈ alkylcarbonyl, arylC₁₋₈ alkoxycarbonyl, aminocarbonyl and C₁₋₈alkylsulfonyl.

The present invention provides in another embodiment, a compoundwherein: D¹ is cycloalkyl, optionally substituted with from 1 to 4substituents independently selected from the group consisting of: C₁₋₈alkyl, amino, hydroxy, C₁₋₈alkylcarbonyl, aminocarbonyl,C₁₋₈alkoxycarbonylamino, aryl C₁₋₈alkoxycarbonylamino, phenyl andheterocyclylC₁₋₈alkylene.

The present invention provides in another embodiment, a compoundwherein: D¹ is cyclopropyl.

The present invention provides in another embodiment, a compoundwherein: D¹ is cyclobutyl.

The present invention provides in another embodiment, a compoundwherein: D¹ is cyclopentyl.

The present invention provides in another embodiment, a compoundwherein: D¹ is phenyl, optionally substituted with from 1 to 4substituents independently selected from the group consisting of:C₁₋₈alkyl, C₁₋₈alkylsulfonyl, C₁₋₈alkylcarbonylheterocyclyl, halo andC₁₋₈alkylcarbonylamino- and C₁₋₈alkoxy.

The present invention provides in another embodiment, a compound wherein—Y¹—R² is selected from the group consisting of:

Y² is N or C;

each R^(4a) is selected from the group consisting of H and halo;

each R^(4c) is independently selected from the group consisting of H andC₁₋₈alkyl;

each R^(4b) is independently selected from the group consisting of H andC₁₋₈alkyl;

X is halo; and

the subscript p is 0, 1, 2 or 3; and

the wavy line indicates the point of attachment to the rest of themolecule.

The present invention provides in another embodiment, a compound whereinthe moiety —Y¹—R² is selected from the group consisting of:

Y² is N or C;

each R^(4a) is selected from the group consisting of H and C₁₋₈alkyl;

each R^(4c) is independently selected from the group consisting of H,C₁₋₈alkyl, C₁₋₈alkoxyC₁₋₈alkyl, aminocarbonyl-, hydroxyl, oxo,C₁₋₈alkoxy and halo;

each R^(4b) is independently selected from the group consisting of H,C₁₋₈alkyl, amino, C₁₋₈alkoxy and heterocyclyl;

X is halo; and

-   -   the subscript p is 0, 1, 2 or 3; and the wavy line indicates the        point of attachment to the rest of the molecule.

The present invention provides in another embodiment, a compoundwherein, D¹ is heteroaryl; optionally substituted with from 1 to 4substituents independently selected from the group consisting of:C₁₋₈alkyl, C₁₋₈alkylcarbonyl, aminocarbonyl, C₁₋₈alkoxycarbonyl, amino,C₁₋₈ alkoxycarbonylamino, arylC₁₋₈alkoxycarbonylamino, hydroxyl, C₁₋₈alkoxy, C₁₋₈alkylsulfonyl, oxo, halo, phenyl andheterocyclylC₁₋₈alkylene.

The present invention provides in another embodiment, a compound whereinD¹ is bicyclic heteroaryl; optionally substituted with from 1 to 4substituents independently selected from the group consisting of:C₁₋₈alkyl, C₁₋₈alkylcarbonyl, aminocarbonyl, C₁₋₈alkoxycarbonyl, amino,C₁₋₈ alkoxycarbonylamino, arylC₁₋₈alkoxycarbonylamino, hydroxyl, C₁₋₈alkoxy, C₁₋₈alkylsulfonyl, oxo, halo, phenyl andheterocyclylC₁₋₈alkylene.

The present invention provides in another embodiment, a compound whereinD¹- is selected from the group consisting of:

-   -   each of which is optionally substituted with from 1 to 4        substituents independently selected from the group consisting        of: C₁₋₈alkyl, C₁₋₈alkylcarbonyl, aminocarbonyl,        C₁₋₈alkoxycarbonyl, amino, C₁₋₈ alkoxycarbonylamino,        arylC₁₋₈alkoxycarbonylamino, hydroxyl, C₁₋₈ alkoxy,        C₁₋₈alkylsulfonyl, oxo, halo, phenyl and        heterocyclylC₁₋₈alkylene; and the wavy line indicates the point        of attachment to the rest of the molecule.

The present invention provides in another embodiment, a compound whereinD¹ is selected from the group consisting of

-   -   each of which is optionally substituted with C₁₋₈alkyl or oxo;        and the wavy line indicates the point of attachment to the rest        of the molecule.

The present invention provides in another embodiment, a compound whereinD¹ is selected from the group consisting of:

and the wavy line indicates the point of attachment to the rest of themolecule.

The present invention provides in another embodiment, a compound whereinD¹- is selected from the group consisting of:

-   -   the wavy line indicates the point of attachment to the rest of        the molecule.

The present invention provides in another embodiment, a compound wherein

D¹- is selected from the group consisting of:

-   -   the wavy line indicates the point of attachment to the rest of        the molecule.

The present invention provides in another embodiment, a compoundwherein,

D¹ is heterocyclyl; optionally substituted with from 1 to 4 substituentsindependently selected from the group consisting of: C₁₋₈alkyl,C₁₋₈alkoxycarbonyl and oxo;

-   -   R¹ is selected from the group consisting of H, C₁₋₈ alkyl,        amino, hydroxyl, C₁₋₈ alkoxy, C₁₋₈ haloalkyl, C₂₋₈ alkenyl, C₂₋₈        alkynyl, oxo, CN, C₁₋₈ alkoxycarbonyl, C₃₋₈ cycloalkyl, aryl and        heterocyclyl; wherein heterocyclyl is optionally substituted        with from 1 to 4 substituents selected from the group consisting        of: alkyl, halo, C₁₋₈ alkylcarbonyl, arylC₁₋₈ alkylenecarbonyl,        aminocarbonyl and C₁₋₈ alkylsulfonyl.

The present invention provides in another group of embodiments, acompound wherein D¹ is selected from the group consisting of:

The present invention provides in another embodiment, a compound whereinD¹ is selected from the group consisting of

-   -   each of which is optionally substituted with from 1 to 3        substituents independently selected from the group consisting        of: C₁₋₈alkyl, C₁₋₈alkoxycarbonyl and oxo; and the wavy line        indicates the point of attachment to the rest of the molecule.

The present invention provides in another embodiment, a compound havingthe formula:

-   -   or a pharmaceutically acceptable salt thereof.

The present invention provides in another embodiment, a compound havingthe formula:

-   -   or a pharmaceutically acceptable salt thereof.

The present invention provides one embodiment, a compound having theformula Ie:

or a pharmaceutically acceptable salt thereof,wherein:

D^(1a) is C₃₋₈cycloalkyl;

R^(1a) is selected from the group consisting H, C₁₋₈alkyl,C₁₋₈alkylaminocarbonyl, C₁₋₈alkylcarbonylamino, aminocarbonyl,carbonylamino, C₃₋₈cycloalkylcarbonylamino, C₃₋₈cycloalkylaminocarbonyl,heteroaryl, heterocyclyl, hydroxyC₁₋₈alkyleneaminocarbonyl;

R^(2a) is selected from the group consisting H, hydroxy, halo,C₁₋₈alkylsulfonyl and heteroaryl; or is combined with R^(1a) and theatoms to which they are attached to form a heterocyclic moiety selectedfrom the group consisting of *—NH—CH═CH— and *—N═CH—CH═N—, wherein *indicates the attachment of R²; and at least one of R^(1a) and R^(2a) isnot H;

each heteraryl or heterocyclyl is independently selected from the groupconsisting of:

each of which is optionally substituted with from 1 to 2 substituentsindependently selected from the group consisting of C₁₋₈alkyl,hydroxyC₁₋₈alkylene, C₁₋₈alkoxyC₁₋₈alkylene, amino, aminocarbonyl,C₁₋₈alkylaminocarbonyl, C₁₋₈alkylcarbonyl, carboxy, hydroxy, halo andC₁₋₈alkylsulfonyl; and the wavy line indicates the point of attachmentto the rest of the molecule.

The present invention provides in another embodiment, a compound havingthe formula If:

or a pharmaceutically acceptable salt thereof,wherein:

D^(1b) is C₃₋₈cycloalkyl;

R^(1b) is selected from the group consisting of heteroaryl,heterocyclyl, heterocyclylcarbonyl and heterocyclylsulfonyl;

R^(2b) is selected from the group consisting of H or halo;

each heteraryl or heterocyclyl is independently selected from the groupconsisting of:

-   -   each of which is substituted with from 1 to 2 substituents        independently selected from the group consisting of hydroxyl,        hydroxyC₁₋₈alkylene, C₁₋₈alkoxyC₈alkylene, C₁₋₈alkylcarbonyl,        C₁₋₈alkylaminocarbonyl, carboxy and C₁₋₈alkylsulfonylamino; and        the wavy line indicates the point of attachment to the rest of        the molecule.

The present invention provides in another embodiment, a compound havingthe formula Ig:

or a pharmaceutically acceptable salt thereof,wherein:

D^(1c) is C₃₋₈cycloalkyl; and

E^(1c) is selected from the group consisting of:

and the wavy line indicates the point of attachment to the rest of themolecule.

The present invention provides in another embodiment, a compound whereinR^(1b) is heterocyclyl, substituted with from 1 to 2 substituentsindependently selected from the group consisting of aminocarbonyl andC₁₋₈alkylsulfonyl.

The present invention provides in another embodiment, a compound whereinD^(1a), D^(1b) or D^(1c) is cyclopropyl or cyclobutyl. The presentinvention provides in another embodiment, a compound wherein D^(1a),D^(1b) or D^(1c) is cyclopropyl. The present invention provides inanother embodiment, a compound wherein D^(1a), D^(1b) or D^(1c) iscyclobutyl.

The present invention provides in another embodiment, a compound havingthe formula:

-   -   or a pharmaceutically acceptable salt thereof.

The present invention provides in one embodiment, a compound having theformula Ih:

or a pharmaceutically acceptable salt thereof,wherein:

D¹ is C₃₋₈cycloalkyl;

R¹ is selected from the group consisting H, amino,C₁₋₈alkoxycarbonylamino, C₁₋₈alkylcarbonylamino, C₁₋₈alkoxy,aminocarbonyl, heteroaryl, heterocyclyl, C₁₋₈alkylthio,C₁₋₈alkylsulfonyl and heterocyclylC₁₋₈alkylene; wherein each heteroarylor heterocyclyl is independently selected from the group consisting of:

each of which is optionally substituted with from 1 to 2 substituentsindependently selected from the group consisting of C₁₋₈alkyl, —CONH₂,hydroxy, halo, C₁₋₈alkylsulfonyl and heteroaryl; and

R² is selected from the group consisting H, C₁₋₈alkyl, halo, C₁₋₈alkoxyand heteroaryl; or is combined with R¹ and the atoms to which they areattached to form a heterocyclic moiety selected from the groupconsisting of —NH—N═CH—*, —CH═N—NH—*, —O—CH₂—CH₂—O—*; —NH—CH═CH—*,—N═CH—CH═CH—*, wherein * indicates the attachment of R² and the wavyline indicates the point of attachment to the rest of the molecule.

The present invention provides in another embodiment, a compound havingthe formula II:

or a pharmaceutically acceptable salt thereof,wherein:

-   -   D¹ is C₃₋₈cycloalkyl;    -   E¹ is selected from the group consisting of:

and the wavy line indicates the point of attachment to the rest of themolecule.

The present invention provides in another embodiment a compound whereinR¹ is

optionally substituted with CONH₂ or C₁₋₈alkylsulfonyl; and the wavyline indicates the point of attachment to the rest of the molecule.

The present invention provides in another embodiment a compound whereinR¹ is C₁₋₈alkylsulfonyl.

The present invention provides in another embodiment a compound whereinR² is H.

The present invention provides in another embodiment a compound havingthe formula Ij:

or a pharmaceutically acceptable salt thereof,wherein: D¹ is C₃₋₈cycloalkyl.

The present invention provides in another embodiment a compound havingthe formula:

or a pharmaceutically acceptable salt thereof, wherein: D¹ isC₃₋₈cycloalkyl.

The present invention provides in another embodiment a compound havingthe formula:

or a pharmaceutically acceptable salt thereof, wherein: D¹ isC₃₋₈cycloalkyl.

The present invention provides in another embodiment a compound whereinD¹ is cyclopropyl or cyclobutyl.

The present invention provides in another embodiment a compound whereinD¹ is cyclopropyl.

The present invention provides in another embodiment a compound whereinD¹ is cyclobutyl.

The present invention provides in another embodiment a compound selectedfrom the group consisting of:

The present invention provides in another embodiment, a compound whereinthe compound is found in the Examples.

The present invention provides in another embodiment, a compound havingthe structure found in the tables.

It is understood that in another group of embodiments, any of the aboveembodiments may also be combined with other embodiments listed herein,to form other embodiments of the invention.

b. Methods of Synthesis

The compounds of the present invention may be prepared by known organicsynthesis techniques, including the methods described in more detail inthe Examples. In general, the compounds of structure (I) above may bemade by the following FIG. 3, wherein all substituents are as definedabove unless indicated otherwise.

Compounds having formula I may be prepared according to FIG. 3.Carboxylic acid 1.1 is converted to acid chloride 1.2 via a one-stepprocedure by treatment with a chlorination agent, such as thionylchloride, and esterification with an alcohol, such as ethanol, to formcompound 1.3 using conditions similar to that described below. Ester 1.3is dichlorinated with a chlorinating agent, such as phosphorousoxychloride. Selective displacement of the 4-chloro group of the2,4-dichloropyrimidine by an appropriate amine, such as R¹-D¹-NH₂(available commercially or synthesized using methods known to thoseskilled in the art), under basic conditions, such as withdiisopropylamine (DIA), provides compounds of formula 1.5. Subsequenthydrolysis of the ester, displacement of the second chloro group withEDC and treatment with ammonia gives compound 1.7. Benzotriazolyl ethercompound 1.7 may also be prepared through a linear route. Displacementof the benzotriazolyl ether group with an appropriate amine, such asR²—Y¹—NH₂ (available commercially or synthesized using methods known tothose skilled in the art), gives the desired product I, wherein R¹ andR² are as previously defined.

One skilled in the art will recognize that in certain embodiments ofstructure (I) when R¹-D¹ or R²—Y¹ comprises a terminal heteroatom, itmay be advantageous to use a protecting group strategy. The protectinggroup can be removed using methods known to those skilled in the art toyield compounds of structure (1).

The compounds of the present invention may generally be utilized as thefree base. Alternatively, the compounds of this invention may be used inthe form of acid addition salts as described below.

c. Inhibition of syk and JAK Kinases

The activity of a specified compound as an inhibitor of a JAK kinase maybe assessed in vitro or in vivo. In some embodiments, the activity of aspecified compound can be tested in a cellular assay. Selectivity couldalso be ascertained in biochemical assays with isolated kinases.

Similar types of assays can be used to assess JAK kinase inhibitoryactivity and to determine the degree of selectivity of the particularcompound as compared to syk kinase. One means of assaying for suchinhibition is detection of the effect of the compounds of the presentinvention on the upregulation of downstream gene products. In theRamos/IL4 assay, B-cells are stimulated with the cytokine Interleukin-4(IL-4) leading to the activation of the JAK/Stat pathway throughphosphorylation of the JAK family kinases, JAK1 and JAK3, which in turnphosphorylate and activate the transcription factor Stat-6. One of thegenes upregulated by activated Stat-6 is the low affinity IgE receptor,CD23. To study the effect of inhibitors (e.g., the 2,4-substitutedpyrimindinediamine compounds described herein) on the JAK1 and JAK3kinases, human Ramos B-cells are stimulated with human IL-4. 10′post-stimulation, cells are subjected to intracellular flow cytometry tomeasure the extent of STAT-6 phosphorylation. 20 to 24 hourspost-stimulation, cells are stained for upregulation of CD23 andanalyzed using flow cytometry. A reduction of the amount ofphosphorylated STAT-6 and/or cell surface CD23 present compared tocontrol conditions indicates that the test compound actively inhibitsthe JAK kinase pathway.

Additionally, IL-6 stimulation of Ramos B-cells induces JAKs 1, 2, andTyk2, leading to Stat-3 and Erk phosphorylation. 10′ post-stimulation,cells are subjected to intracellular flow cytometry to measure theability of compound to inhibit these phosphorylation events. Tospecifically measure the activity of JAK2, the CellSensor irf1-bla HELcell line expressing the beta-lactamase reporter gene controlled byStat5 will be used (Invitrogen, Carlsbad, Calif.). These cells express aconstitutively active JAK2 mutant (JAK2V617F), found naturally inmyeloproliferative neoplasms (Constantinescu, S., et. al, Trends BiochemSci., 2008; 33:122-31). A reduction in the amount of beta-lactamasereporter gene expression is used a measure of the JAK2 inhibitoryactivity of compounds.

The activity of the compounds of the invention may additionally becharacterized by assaying the effect of the compounds of the presentinvention described herein on A549 lung epithelial cells and U937 cells.A549 lung epithelial cells and U937 cells up-regulate ICAM-1 (CD54)surface expression in response to a variety of different stimuli.Therefore, using ICAM-1 expression as readout, test compound effects ondifferent signaling pathways can be assessed in the same cell type.Stimulation with IL-1β through the IL-1 β receptor activates theTRAF6/NFκB pathway resulting in up-regulation of ICAM-1. IFN.gamma.induces ICAM-1 up-regulation through activation of the JAK1/JAK2pathway. The up-regulation of ICAM-1 can be quantified by flow cytometryacross a compound dose curve and EC₅₀ values are calculated.

The activity of the compounds of the invention may additionally becharacterized by assaying the effect of the compounds of the presentinvention described herein on A549 lung epithelial cells and U937 cells.A549 lung epithelial cells and U937 cells up-regulate ICAM-1 (CD54)surface expression in response to a variety of different stimuli.Therefore, using ICAM-1 expression as readout, test compound effects ondifferent signaling pathways can be assessed in the same cell type.Stimulation with IL-1β through the IL-1 β receptor activates theTRAF6/NFκB pathway resulting in up-regulation of ICAM-1. IFN.gamma.induces ICAM-1 up-regulation through activation of the JAK1/JAK2pathway. The up-regulation of ICAM-1 can be quantified by flow cytometryacross a compound dose curve and EC₅₀ values are calculated. Exemplaryassays of this type are described in greater detail in the Examples.

Active compounds as described herein generally inhibit the JAK kinasepathway with an IC₅₀ in the range of about 1 mM or less, as measured inthe assays described herein. Of course, skilled artisans will appreciatethat compounds which exhibit lower IC₅₀s, (on the order, for example, of100 μM, 75 μM, 50 μM, 40 μM, 30 μM, 20 μM, 15 μM, 10 μM, 5 μM, 1 μM, 500nM, 100 nM, 10 nM, 1 nM, or even lower) can be particularly useful intherapeutic applications. In instances where activity specific to aparticular cell type is desired, the compound can be assayed foractivity with the desired cell type and counter-screened for a lack ofactivity against other cell types. The desired degree of “inactivity” insuch counter screens, or the desired ratio of activity vs. inactivity,may vary for different situations and can be selected by the user.

The active compounds also typically inhibit IL-4 stimulated expressionof CD23 in B-cells with an IC₅₀ in the range of about 20 μM or less,typically in the range of about 10 μM, 1 μM, 500 nM, 100 nM, 10 nM, 1nM, or even lower. A suitable assay that can be used is the assaydescribed in the Examples, “Assay for Ramos B-cell Line Stimulated withIL-4.” In certain embodiments, the active compounds of the presentinvention have an IC₅₀ of less than or equal to 5 μM, greater than 5 μMbut less than 20 μM, greater than 20 μM, or greater than 20 μM but lessthan 50 μM in the assay described in the Examples.

The active compounds also typically inhibit expression of ICAM1 (CD54)induced by IFN.gamma. exposure in U937 or A549 cells with an IC₅₀ in therange of about 20 μM or less, typically in the range of about 10 μM, 1μM, 500 nM, 100 nM, 10 nM, 1 nM, or even lower. The IC₅₀ againstexpression of ICAM (CD54) in IFN.gamma. stimulated cells can bedetermined in a functional cellular assay with an isolated A549 or U937cell line. Suitable assays that can be used are the assays described inthe Examples, “A549 Epithelial Line Stimulated with IFNγ” and “U937IFN.gamma. ICAM1 FACS Assay,” respectively. In certain embodiments, theactive compounds of the present invention have an IC₅₀ of less than orequal to 20 μM, greater than 20 μM, or greater than 20 μM but less than50 μM in the assays described in the Examples.

d. Compositions and Methods of Administration

The present invention further provides compositions comprising one ormore compounds of formula (I) or a pharmaceutically acceptable salt,ester or prodrug thereof, and a pharmaceutically acceptable carrier ordiluent. It will be appreciated that the compounds of formula (I)) inthis invention may be derivatized at functional groups to provideprodrug derivatives which are capable of conversion back to the parentcompounds in vivo. Examples of such prodrugs include the physiologicallyacceptable and metabolically labile ester derivatives, such asmethoxymethyl esters, methylthiomethyl esters, or pivaloyloxymethylesters derived from a hydroxyl group of the compound or a carbamoylmoiety derived from an amino group of the compound. Additionally, anyphysiologically acceptable equivalents of the compounds of formula (I),similar to metabolically labile esters or carbamates, which are capableof producing the parent compounds of formula (I) in vivo, are within thescope of this invention.

As used herein, the term “pharmaceutically acceptable salts” refers toany acid or base addition salt whose counter-ions are non-toxic to thepatient in pharmaceutical doses of the salts. A host of pharmaceuticallyacceptable salts are well known in the pharmaceutical field. Ifpharmaceutically acceptable salts of the compounds of this invention areutilized in these compositions, those salts are preferably derived frominorganic or organic acids and bases. Included among such acid salts arethe following: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphor sulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, lucoheptanoate, glycerophosphate, hemisulfate, heptanoate,hexanoate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate,persulfate, 3-phenyl-propionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate, undecanoate, hydrohalides(e.g., hydrochlorides and hydrobromides), sulphates, phosphates,nitrates, sulphamates, malonates, salicylates,methylene-bis-b-hydroxynaphthoates, gentisates, isethionates,di-p-toluoyltartrates, ethanesulphonates, cyclohexylsulphamates,quinates, and the like. Pharmaceutically acceptable base addition saltsinclude, without limitation, those derived from alkali or alkaline earthmetal bases or conventional organic bases, such as triethylamine,pyridine, piperidine, morpholine, N-methylmorpholine, ammonium salts,alkali metal salts, such as sodium and potassium salts, alkaline earthmetal salts, such as calcium and magnesium salts, salts with organicbases, such as dicyclohexylamine salts, N-methyl-D-glucamine, and saltswith amino acids such as arginine, lysine, and so forth.

Furthermore, the basic nitrogen-containing groups may be quaternizedwith agents like lower alkyl halides, such as methyl, ethyl, propyl andbutyl chlorides, bromides and iodides; dialkyl sulfates, such asdimethyl, diethyl, dibutyl and diamyl sulfates, long chain halides, suchas decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides;aralkyl halides, such as benzyl and phenethyl bromides and others. Wateror oil-soluble or dispersible products are thereby obtained.

The compounds utilized in the compositions and methods of this inventionmay also be modified by appending appropriate functionalities to enhanceselective biological properties. Such modifications are known in the artand include those which increase biological penetration into a givenbiological system (e.g., blood, lymphatic system, central nervoussystem, etc.), increase oral availability, increase solubility to allowadministration by injection, alter metabolism and alter rate ofexcretion.

The pharmaceutical compositions of the invention can be manufactured bymethods well known in the art such as conventional granulating, mixing,dissolving, encapsulating, lyophilizing, or emulsifying processes, amongothers. Compositions may be produced in various forms, includinggranules, precipitates, or particulates, powders, including freezedried, rotary dried or spray dried powders, amorphous powders, tablets,capsules, syrup, suppositories, injections, emulsions, elixirs,suspensions or solutions. Formulations may optionally containstabilizers, pH modifiers, surfactants, bioavailability modifiers andcombinations of these.

The term “unit dosage form” refers to physically discrete units suitableas unitary dosages for human subjects and other mammals, each unitcontaining a predetermined quantity of drug calculated to produce thedesired onset, tolerability, and/or therapeutic effects, in associationwith a suitable pharmaceutical excipient (e.g., an ampoule). Inaddition, more concentrated compositions may be prepared, from which themore dilute unit dosage compositions may then be produced. The moreconcentrated compositions thus will contain substantially more than,e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times the amountof one or more syk and/or JAK inhibitors.

Methods for preparing such dosage forms are known to those skilled inthe art (see, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 18THED., Mack Publishing Co., Easton, Pa. (1990)). In addition,pharmaceutically acceptable salts of the syk and/or JAK inhibitors ofthe present invention (e.g., acid addition salts) may be prepared andincluded in the compositions using standard procedures known to thoseskilled in the art of synthetic organic chemistry and described, e.g.,by J. March, Advanced Organic Chemistry: Reactions, Mechanisms andStructure, 4^(th) Ed. (New York: Wiley-Interscience, 1992).

The compositions typically include a conventional pharmaceutical carrieror excipient and may additionally include other medicinal agents,carriers, adjuvants, diluents, tissue permeation enhancers,solubilizers, and the like. Preferably, the composition will containabout 0.01% to about 90%, preferably about 0.1% to about 75%, morepreferably about 0.1% to 50%, still more preferably about 0.1% to 10% byweight of one or more syk and/or JAK inhibitors, with the remainderconsisting of suitable pharmaceutical carrier and/or excipients.Appropriate excipients can be tailored to the particular composition androute of administration by methods well known in the art, e.g.,REMINGTON'S PHARMACEUTICAL SCIENCES, supra.

Pharmaceutically acceptable carriers that may be used in thesecompositions include ion exchangers, alumina, aluminum stearate,lecithin, serum proteins, such as human serum albumin, buffersubstances, such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

Examples of suitable excipients include, but are not limited to,lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,calcium phosphate, alginates, tragacanth, gelatin, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water,saline, syrup, methylcellulose, ethylcellulose,hydroxypropylmethylcellulose, and polyacrylic acids such as Carbopols.The compositions can additionally include lubricating agents such astalc, magnesium stearate, and mineral oil; wetting agents; emulsifyingagents; suspending agents; preserving agents such as methyl-, ethyl-,and propyl-hydroxy-benzoates; pH adjusting agents such as inorganic andorganic acids and bases; sweetening agents; and flavoring agents.

Administration of a composition comprising one or more syk and/or JAKinhibitors with one or more suitable pharmaceutical excipients asadvantageous can be carried out via any of the accepted modes ofadministration. Thus, administration can be, for example, oral, topical,intravenous, subcutaneous, transcutaneous, transdermal, intramuscular,intra-joint, parenteral, intra-arteriole, intradermal, intraventricular,intracranial, intraperitoneal, intralesional, intranasal, rectal,vaginal, by inhalation or via an implanted reservoir. The term“parenteral” as used herein includes subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional and intracranial injection orinfusion techniques. Preferably, the compositions are administeredorally or intravenously. The formulations of the invention may bedesigned as short-acting, fast-releasing, or long-acting. Still further,compounds can be administered in a local rather than systemic means,such as administration (e.g., injection) as a sustained releaseformulation. According to a representative embodiment, the compositionsof this invention are formulated for pharmaceutical administration to amammal, preferably a human being.

The compositions of the present invention containing one or more sykand/or JAK inhibitors can be administered repeatedly, e.g., at least 2,3, 4, 5, 6, 7, 8, or more times, or the composition may be administeredby continuous infusion. Suitable sites of administration include, butare not limited to, skin, bronchial, gastrointestinal, anal, vaginal,eye, and ear. The formulations may take the form of solid, semi-solid,lyophilized powder, or liquid dosage forms, such as, for example,tablets, pills, capsules, powders, solutions, suspensions, emulsions,suppositories, retention enemas, creams, ointments, lotions, gels,aerosols, or the like, preferably in unit dosage forms suitable forsimple administration of precise dosages.

The pharmaceutical compositions of this invention may be in any orallyacceptable dosage form, including tablets, capsules, cachets, emulsions,suspensions, solutions, syrups, elixirs, sprays, boluses, lozenges,powders, granules, and sustained-release formulations. Suitableexcipients for oral administration include pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, andthe like. In the case of tablets for oral use, carriers that arecommonly used include lactose and corn starch. Lubricating agents, suchas magnesium stearate, are also typically added. For a capsule form,useful diluents include lactose and dried cornstarch. When aqueoussuspensions are required for oral use, the active ingredient is combinedwith emulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

In some embodiments, the compositions take the form of a pill, tablet,or capsule, and thus, the composition can contain, along with one ormore syk and/or JAK inhibitors, a diluent such as lactose, sucrose,dicalcium phosphate, and the like; a disintegrant such as starch orderivatives thereof; a lubricant such as magnesium stearate and thelike; and/or a binder such a starch, gum acacia, polyvinylpyrrolidone,gelatin, cellulose and derivatives thereof. A tablet can be made by anycompression or molding process known to those of skill in the art.Compressed tablets may be prepared by compressing in a suitable machinethe syk and/or JAK inhibitors in a free-flowing form, e.g., a powder orgranules, optionally mixed with accessory ingredients, e.g., binders,lubricants, diluents, disintegrants, or dispersing agents. Moldedtablets can be made by molding in a suitable machine a mixture of thepowdered syk and/or JAK inhibitors with any suitable carrier.

Alternatively, the pharmaceutical compositions of this invention may bein the form of suppositories for rectal administration. These may beprepared by mixing the agent with a suitable non-irritating excipientwhich is solid at room temperature but liquid at rectal temperature andtherefore will melt in the rectum to release the drug. Such materialsinclude cocoa butter, beeswax, polyethylene glycol (PEG), hard fat,and/or hydrogenated cocoglyceride. Compositions suitable for rectaladministration may also comprise a rectal enema unit containing one ormore syk and/or JAK inhibitors and pharmaceutically-acceptable vehicles(e.g., 50% aqueous ethanol or an aqueous salt solution) that arephysiologically compatible with the rectum and/or colon. The rectalenema unit contains an applicator tip protected by an inert cover,preferably comprised of polyethylene, lubricated with a lubricant suchas white petrolatum, and preferably protected by a one-way valve toprevent back-flow of the dispensed formula. The rectal enema unit isalso of sufficient length, preferably two inches, to be inserted intothe colon via the anus.

Liquid compositions can be prepared by dissolving or dispersing one ormore syk and/or JAK inhibitors and optionally one or morepharmaceutically acceptable adjuvants in a carrier such as, for example,aqueous saline, aqueous dextrose, glycerol, ethanol, and the like, toform a solution or suspension, e.g., for oral, topical, or intravenousadministration. Pharmaceutical formulations may be prepared as liquidsuspensions or solutions using a sterile liquid, such as oil, water,alcohol, and combinations thereof. Pharmaceutically suitablesurfactants, suspending agents or emulsifying agents, may be added fororal or parenteral administration. Suspensions may include oils, such aspeanut oil, sesame oil, cottonseed oil, corn oil and olive oil.Suspension preparation may also contain esters of fatty acids, such asethyl oleate, isopropyl myristate, fatty acid glycerides and acetylatedfatty acid glycerides. Suspension formulations may include alcohols,such as ethanol, isopropyl alcohol, hexadecyl alcohol, glycerol andpropylene glycol. Ethers, such as poly(ethyleneglycol), petroleumhydrocarbons, such as mineral oil and petrolatum, and water may also beused in suspension formulations.

The pharmaceutical compositions of this invention may also be in atopical form, especially when the target of treatment includes areas ororgans readily accessible by topical application, including diseases ofthe eye, the skin, or the lower intestinal tract. Suitable topicalformulations are readily prepared for each of these areas or organs. Fortopical administration, the composition containing one or more sykand/or JAK inhibitors can be in the form of emulsions, lotions, gels,foams, creams, jellies, solutions, suspensions, ointments, andtransdermal patches.

Topical application for the lower intestinal tract may be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used. For topicalapplications, the pharmaceutical compositions may be formulated in asuitable ointment containing the active component suspended or dissolvedin one or more carriers. Carriers for topical administration of thecompounds of this invention include, but are not limited to, mineraloil, liquid petrolatum, white petrolatum, propylene glycol,polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.Alternatively, the pharmaceutical compositions may be formulated in asuitable lotion or cream containing the active components suspended ordissolved in one or more pharmaceutically acceptable carriers. Suitablecarriers include mineral oil, sorbitan monostearate, polysorbate 60,cetyl esters, wax, cetyl alcohol, 2-octyldodecanol, benzyl alcohol andwater.

The pharmaceutical compositions of this invention may also beadministered by nasal aerosol or inhalation. For delivery by inhalation,the compositions can be delivered as a dry powder or in liquid form viaa nebulizer. Such compositions are prepared according to techniquesknown in the art of pharmaceutical formulation and may be prepared assolutions in saline, employing benzyl alcohol or other suitablepreservatives, absorption promoters to enhance bioavailability,fluorocarbons and/or other conventional solubilizing or dispersingagents.

For ophthalmic use, the pharmaceutical compositions may be formulated asmicronized suspensions in isotonic, pH adjusted sterile saline, or,preferably, as solutions in isotonic, pH adjusted sterile saline, eitherwith our without a preservative, such as benzylalkonium chloride.Alternatively, for ophthalmic uses, the pharmaceutical compositions maybe formulated in an ointment, such as petrolatum.

For parenteral administration, the compositions can be in the form ofsterile injectable solutions and sterile packaged powders. Preferably,injectable solutions are formulated at a pH of about 4.5 to about 7.5.

Sterile injectable forms of the compositions of this invention may beaqueous or oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or di-glycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such ascarboxymethyl cellulose or similar dispersing agents which are commonlyused in the formulation of pharmaceutically acceptable dosage formsincluding emulsions and suspensions. Other commonly used surfactants,such as Tweens, Spans and other emulsifying agents or bioavailabilityenhancers which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms may also be used for thepurposes of formulation. Compounds may be formulated for parenteraladministration by injection such as by bolus injection or continuousinfusion. A unit dosage form for injection may be in ampoules or inmulti-dose containers.

The compositions of the present invention can also be provided in alyophilized form. Such compositions may include a buffer, e.g.,bicarbonate, for reconstitution prior to administration, or the buffermay be included in the lyophilized composition for reconstitution with,e.g., water. The lyophilized composition may further comprise a suitablevasoconstrictor, e.g., epinephrine. The lyophilized composition can beprovided in a syringe, optionally packaged in combination with thebuffer for reconstitution, such that the reconstituted composition canbe immediately administered to a patient.

Any of the above dosage forms containing effective amounts are withinthe bounds of routine experimentation and within the scope of theinvention. A therapeutically effective dose may vary depending upon theroute of administration and dosage form. The representative compound orcompounds of the invention is a formulation that exhibits a hightherapeutic index. The therapeutic index is the dose ratio between toxicand therapeutic effects which can be expressed as the ratio between LD₅₀and ED₅₀. The LD₅₀ is the dose lethal to 50% of the population and theED₅₀ is the dose therapeutically effective in 50% of the population. TheLD₅₀ and ED₅₀ are determined by standard pharmaceutical procedures inanimal cell cultures or experimental animals.

Besides those representative dosage forms described above,pharmaceutically acceptable excipients and carriers and dosage forms aregenerally known to those skilled in the art and are included in theinvention. It should be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex and diet of the patient, and thetime of administration, rate of excretion, drug combination, judgment ofthe treating physician and severity of the particular disease beingtreated. The amount of active ingredient(s) will also depend upon theparticular compound and other therapeutic agent, if present, in thecomposition.

e. Methods of Use

The invention provides methods of inhibiting or decreasing syk and/orJAK activity as well as treating or ameliorating a syk and/or JAKassociated state, symptom, condition, disorder or disease in a patientin need thereof (e.g., human or non-human). In one embodiment, the sykand/or JAK associated state, symptom, condition, disorder or disease ismediated, at least in part by syk and/or JAK kinase activity. In morespecific embodiments, the present invention provides a method fortreating a condition or disorder mediated at least in part by syk and/orJAK kinase activity is cardiovascular disease, inflammatory disease orautoimmune disease.

In one embodiment, the invention provides methods for preventing ortreating a condition in a mammal characterized by undesired thrombosiscomprising the step of administering to the mammal a therapeuticallyeffective amount of a compound of the present invention. Such conditionsinclude, but are not limited, to restenosis, acute coronary syndrome,myocardial infarction, unstable angina, refractory angina, occlusivecoronary thrombosis occurring post-thrombolytic therapy or post-coronaryangioplasty, a thrombotically mediated cerebrovascular syndrome, embolicstroke, thrombotic stroke, transient ischemic attacks, venousthrombosis, deep venous thrombosis, pulmonary embolism, coagulopathy,disseminated intravascular coagulation, thrombotic thrombocytopenicpurpura, thromboangiitis obliterans, thrombotic disease associated withheparin-induced thrombocytopenia, thrombotic complications associatedwith extracorporeal circulation, thrombotic complications associatedwith instrumentation such as cardiac or other intravascularcatheterization, intra-aortic balloon pump, coronary stent or cardiacvalve, conditions requiring the fitting of prosthetic devices, and thelike.

In a further embodiment, the present invention provides a method fortreating thrombosis, immune thrombocytic purura, heparin inducedthrombocytopenia, dilated cardiomypathy, sickle cell disease,atherosclerosis, myocardial infarction, vacular inflammation, unstableangina or acute coronary syndromes.

In another embodiment, the present invention also provides a method fortreating allergy, asthma, theumatoid arthritis, B Cell mediated diseasesuch as Non-Hodgkin's Lymphoma, anti phospholipids syndrome, lupus,psoriasis, multiple sclerosis, end stage renal disease or chroniclymphocytic leukemia.

In another embodiment, the present invention provides a method fortreating hemolytic anemia or immune thrombocytopenic purpura.

The compounds described herein are also potent and/or selectiveinhibitors of JAK kinases. As a consequence of this activity, thecompounds can be used in a variety of in vitro, in vivo, and ex vivocontexts to regulate or inhibit JAK kinase activity, signaling cascadesin which JAK kinases play a role, and the biological responses effectedby such signaling cascades. For example, in one embodiment, thecompounds can be used to inhibit JAK kinase, either in vitro or in vivo,in virtually any cell type expressing the JAK kinase, such as inhematopoietic cells in which, for example, JAK3 is predominantlyexpressed. They may also be used to regulate signal transductioncascades in which JAK kinases, particularly JAK3, play a role. SuchJAK-dependent signal transduction cascades include, but are not limitedto, the signaling cascades of cytokine receptors that involve the commongamma chain, such as, for example, the IL-4, IL-7, IL-5, IL-9, IL-15 andIL-21, or IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 receptor signalingcascades. The compounds may also be used in vitro or in vivo toregulate, and in particular to inhibit, cellular or biological responsesaffected by such JAK-dependent signal transduction cascades. Suchcellular or biological responses include, but are not limited to,IL-4/ramos CD23 upregulation and IL-2 mediated T-cell proliferation.Importantly, the compounds can be used to inhibit JAK kinases in vivo asa therapeutic approach towards the treatment or prevention of diseasesmediated, either wholly or in part, by a JAK kinase activity (referredto herein as “JAK kinase mediated diseases”). Non-limiting examples ofJAK kinase mediated diseases that can be treated or prevented with thecompounds include, but are not limited to, the following: allergies;asthma; autoimmune diseases such as transplant rejection (e.g., kidney,heart, lung, liver, pancreas, skin, small intestine, large intestine,host versus graft reaction (HVGR), and graft versus host reaction(GVHR)), rheumatoid arthritis, and amyotrophic lateral sclerosis; T-cellmediated autoimmune diseases such as multiple sclerosis, psoraiasis, andSjogren's syndrome; Type II inflammatory diseases such as vascularinflammation (including vasculitis, arteritis, atherosclerosis, andcoronary artery disease); diseases of the central nervous system such asstroke; pulmonary diseases such as bronchitis obliteraus and primarypulmonary hypertension; solid, delayed Type IV hypersensitivityreactions; and hematologic malignancies such as leukemia and lymphomas.

Examples of diseases that are mediated, at least in part, by JAK kinasesthat can be treated or prevented according to the methods include, butare not limited to, allergies, asthma, autoimmune diseases such astransplant rejection (e.g., kidney, heart, lung, liver, pancreas, skin,host versus graft reaction (HVGR), etc.), rheumatoid arthritis, andamyotrophic lateral sclerosis, multiple sclerosis, psoraiasis andSjogren's syndrome, Type II inflammatory disease such as vascularinflammation (including vasculitis, arteritis, atherosclerosis andcoronary artery disease) or other inflammatory diseases such asosteoarthritis, inflammatory bowel disease, ulcerative colitis, Crohn'sdisease, idiopathic inflammatory bowel disease, irritable bowelsyndrome, spastic colon, low grade scarring (e.g., scleroderma,increased fibrosis, keloids, post-surgical scars, pulmonary fibrosis,vascular spasms, migraine, reperfusion injury and post myocardialinfarction), and sicca complex or syndrome, diseases of the centralnervous system such as stroke, pulmonary diseases such as bronchitisobliterous and primary and primary pulmonary hypertension, delayed orcell-mediated, Type IV hypersensitivity and solid and hematologicmalignancies such as leukemias and lymphomas.

In another embodiment, this invention provides a method of inhibiting anactivity of a JAK kinase, comprising contacting the JAK kinase with anamount of a compound effective to inhibit an activity of the JAK kinase,wherein the compound is selected from the compounds of this invention.In certain embodiments of the methods described herein, the method iscarried out in vivo.

In another embodiment, this invention provides a method of inhibiting anactivity of a JAK kinase, comprising contacting in vitro a JAK3 kinasewith an amount of a compound effective to inhibit an activity of the JAKkinase, wherein the compound is selected from the compounds of thisinvention.

In a specific embodiment, the compounds can be used to treat and/orprevent rejection in organ and/or tissue transplant recipients (i.e.,treat and/or prevent allorgraft rejection). Allografts can be rejectedthrough either a cell-mediated or humoral immune reaction of therecipient against transplant (histocompability) antigens present on themembranes of the donor's cells. The strongest antigens are governed by acomplex of genetic loci termed human leukocyte group A (HLA) antigens.Together with the ABO blood groups antigens, they are the chieftransplantation antigens detectable in humans.

Rejection following transplantation can generally be broken into threecategories: hyperacute, occurring hours to days followingtransplantation; acute, occurring days to months followingtransplantation; and chronic, occurring months to years followingtransplantation.

Hyperacute rejection is caused mainly by the production of hostantibodies that attack the graft tissue. In a hyperacute rejectionreaction, antibodies are observed in the transplant vascular very soonafter transplantation. Shortly thereafter, vascular clotting occurs,leading to ischemia, eventual necrosis and death. The graft infarctionis unresponsive to known immunosuppressive therapies. Because HLAantigens can be identified in vitro, pre-transplant screening is used tosignificantly reduce hyperacute rejection. As a consequence of thisscreening, hyperacute rejection is relatively uncommon today.

Acute rejection is thought to be mediated by the accumulation of antigenspecific cells in the graft tissue. The T-cell-mediated immune reactionagainst these antigens (i.e., HVGR or GVHR) is the principle mechanismof acute rejection. Accumulation of these cells leads to damage of thegraft tissue. It is believed that both CD4+ helper T-cells and CD8+cytotoxic T-cells are involved in the process and that the antigen ispresented by donor and host dendritic cells. The CD4+ helper T-cellshelp recruit other effector cells, such as macrophapges and eosinophils,to the graft. Accessing T-cell activation signal transduction cascades(for example, CD28, CD40L, and CD2 cascades) are also involved.

The cell-mediated acute rejection can be reversed in many cases byintensifying immunotherapy. After successful reversal, severely damagedelements of the graft heal by fibrosis and the remainder of the graftappears normal. After resolution of acute rejection, dosages ofimmunosuppressive drugs can be reduced to very low levels.

Chronic rejection, which is a particular problem in renal transplants,often progresses insidiously despite increased immunosuppressivetherapy. It is thought to be due, in large part, to cell-mediated TypeIV hypersensitivity. The pathologic profile differs from that of acuterejection. The arterial endothelium is primarily involved with extensiveproliferation that may gradually occlude the vessel lumen, leading toischemia, fibrosis, a thickened intima, and atherosclerotic changes.Chronic rejection is mainly due to a progressive obliteration of graftvasculature and resembles a slow, vasculitic process.

In Type IV hypersensitivity, CD8 cytotoxic T-cells and CD4 helper Tcells recognize either intracellular or extracellular synthesizedantigen when it is complexed, respectively, with either Class I or ClassII MHC molecules. Macrophages function as antigen-presenting cells andrelease IL-1, which promotes proliferation of helper T-cells. HelperT-cells release interferon gamma and IL-2, which together regulatedelayed hyperactivity reactions mediated by macrophage activation andimmunity mediated by T cells. In the case of organ transplant, thecytotoxic T-cells destroy the graft cells on contact.

Since JAK kinases play a critical role in the activation of T-cells, thecompounds described herein can be used to treat and/or prevent manyaspects of transplant rejection, and are particularly useful in thetreatment and/or prevention of rejection reactions that are mediated, atleast in part, by T-cells, such as HVGR or GVHR. The compounds can alsobe used to treat and/or prevent chronic rejection in transplantrecipients and, in particular, in renal transplant recipients. Thecompound can also be administered to a tissue or an organ prior totransplanting the tissue or organ in the transplant recipient.

In another embodiment, this invention provides a method of treating aT-cell mediated autoimmune disease, comprising administering to apatient suffering from such an autoimmune disease an amount of acompound effective to treat the autoimmune disease wherein the compoundis selected from the compounds of the invention. In certain embodimentsof the methods the autoimmune disease is multiple sclerosis (MS),psoriasis, or Sjogran's syndrome. Such autoimmune disease include, butare not limited to, those autoimmune diseases that are frequentlydesignated as single organ or single cell-type autoimmune disorders andthose autoimmune disease that are frequently designated as involvingsystemic autoimmune disorder. Non-limiting examples of diseasesfrequently designated as single organ or single cell-type autoimmunedisorders include: Hashimoto's thyroiditis, autoimmune hemolytic anemia,autoimmune atrophic gastritis of pernicious anemia, autoimmuneencephalomyelitis, autoimmune orchitis, Goodpasture's disease,autoimmune thrombocytopenia, sympathetic ophthalmia, myasthenia gravis,Graves' disease, primary biliary cirrhosis, chronic aggressivehepatitis, ulcerative colitis and membranous glomerulopathy.Non-limiting examples of diseases often designated as involving systemicautoimmune disorder include: systemic lupus erythematosis, rheumatoidarthritis, Sjogren's syndrome, Reiter's syndrome,polymyositis-dermatomyositis, systemic sclerosis, polyarteritis nodosa,multiple sclerosis and bullous pemphigoid. Additional autoimmunediseases, which can be .beta.-cell (humoral) based or T-cell based,include Cogan's syndrome, ankylosing spondylitis, Wegener'sgranulomatosis, autoimmune alopecia, Type I or juvenile onset diabetes,and thyroiditis.

The types of autoimmune diseases that may be treated or prevented withsuch prodrugs generally include those disorders involving tissue injurythat occurs as a result of a humoral and/or cell-mediated response toimmunogens or antigens of endogenous and/or exogenous origin. Suchdiseases are frequently referred to as diseases involving thenonanaphylactic (i.e., Type II, Type III and/or Type IV)hypersensitivity reactions.

Type I hypersensitivity reactions generally result from the release ofpharmacologically active substances, such as histamine, from mast and/orbasophil cells following contact with a specific exogenous antigen. Asmentioned above, such Type I reactions play a role in numerous diseases,including allergic asthma, allergic rhinitis, etc.

Type II hypersensitivity reactions (also referred to as cytotoxic,cytolytic complement-dependent or cell-stimulating hypersensitivityreactions) result when immunoglobulins react with antigenic componentsof cells or tissue, or with an antigen or hapten that has becomeintimately coupled to cells or tissue. Diseases that are commonlyassociated with Type II hypersensitivity reactions include, but are notlimited, to autoimmune hemolytic anemia, erythroblastosis fetalis andGoodpasture's disease.

Type III hypersensitivity reactions, (also referred to as toxic complex,soluble complex, or immune complex hypersensitivity reactions) resultfrom the deposition of soluble circulating antigen-immunoglobulincomplexes in vessels or in tissues, with accompanying acute inflammatoryreactions at the site of immune complex deposition. Non-limitingexamples of prototypical Type III reaction diseases include the Arthusreaction, rheumatoid arthritis, serum sickness, systemic lupuserythematosis, certain types of glomerulonephritis, multiple sclerosisand bullous pemphingoid.

Type IV hypersensitivity reactions (frequently called cellular,cell-mediated, delayed, or tuberculin-type hypersensitivity reactions)are caused by sensitized T-lymphocytes which result from contact with aspecific antigen. Non-limiting examples of diseases cited as involvingType IV reactions are contact dermatitis and allograft rejection.

Autoimmune diseases associated with any of the above nonanaphylactichypersensitivity reactions may be treated or prevented with the prodrugsaccording to structural formulae (I) and (Ia). In particular, themethods may be used to treat or prevent those autoimmune diseasesfrequently characterized as single organ or single cell-type autoimmunedisorders including, but not limited to: Hashimoto's thyroiditis,autoimmune hemolytic anemia, autoimmune atrophic gastritis of perniciousanemia, autoimmune encephalomyelitis, autoimmune orchitis, Goodpasture'sdisease, autoimmune thrombocytopenia, sympathetic ophthalmia, myastheniagravis, Graves' disease, primary biliary cirrhosis, chronic aggressivehepatitis, ulcerative colitis and membranous glomerulopathy, as well asthose autoimmune diseases frequently characterized as involving systemicautoimmune disorder, which include but are not limited to: systemiclupus erythematosis (SLE), rheumatoid arthritis, Sjogren's syndrome,Reiter's syndrome, polymyositis-dermatomyositis, systemic sclerosis,polyarteritis nodosa, multiple sclerosis and bullous pemphigoid.

It will be appreciated by skilled artisans that many of the above-listedautoimmune diseases are associated with severe symptoms, theamelioration of which provides significant therapeutic benefit even ininstances where the underlying autoimmune disease may not beameliorated.

Therapy using the compounds described herein can be applied alone, or itcan be applied in combination with or adjunctive to other commonimmunosuppressive therapies, such as, for example, the following:mercaptopurine; corticosteroids such as prednisone; methylprednisoloneand prednisolone; alkylating agents such as cyclophosphamide;calcineurin inhibitors such as cyclosporine, sirolimus, and tacrolimus;inhibitors of inosine monophosphate dehydrogenase (IMPDH) such asmycophenolate, mycophenolate mofetil, and azathioprine; and agentsdesigned to suppress cellular immunity while leaving the recipient'shumoral immunologic response intact, including various antibodies (forexample, antilymphocyte globulin (ALG), antithymocyte globulin (ATG),monoclonal anti-T-cell antibodies (OKT3)) and irradiation. These variousagents can be used in accordance with their standard or common dosages,as specified in the prescribing information accompanying commerciallyavailable forms of the drugs (see also: the prescribing information inthe 2006 Edition of The Physician's Desk Reference), the disclosures ofwhich are incorporated herein by reference. Azathioprine is currentlyavailable from Salix Pharmaceuticals, Inc., under the brand name AZASAN;mercaptopurine is currently available from Gate Pharmaceuticals, Inc.,under the brand name PURINETHOL; prednisone and prednisolone arecurrently available from Roxane Laboratories, Inc.; Methyl prednisoloneis currently available from Pfizer; sirolimus (rapamycin) is currentlyavailable from Wyeth-Ayerst under the brand name RAPAMUNE; tacrolimus iscurrently available from Fujisawa under the brand name PROGRAF;cyclosporine is current available from Novartis under the brand dameSANDIMMUNE and from Abbott under the brand name GENGRAF; IMPDHinhibitors such as mycophenolate mofetil and mycophenolic acid arecurrently available from Roche under the brand name CELLCEPT and fromNovartis under the brand name MYFORTIC; azathioprine is currentlyavailable from Glaxo Smith Kline under the brand name IMURAN; andantibodies are currently available from Ortho Biotech under the brandname ORTHOCLONE, from Novartis under the brand name SIMULECT(basiliximab), and from Roche under the brand name ZENAPAX (daclizumab).

In another embodiment, the compounds could be administered either incombination or adjunctively with an inhibitor of a syk kinase. sykkinase is a tyrosine kinase known to play a critical role in Fcyreceptor signaling, as well as in other signaling cascades, such asthose involving B-cell receptor signaling (Turner et al., (2000),Immunology Today 21:148-154) and integrins beta (1), beta (2), and beta(3) in neutrophils (Mocsai et al., (2002), Immunity 16:547-558). Forexample, syk kinase plays a pivotal role in high affinity IgE receptorsignaling in mast cells that leads to activation and subsequent releaseof multiple chemical mediators that trigger allergic attacks. However,unlike the JAK kinases, which help regulate the pathways involved indelayed or cell-mediated Type IV hypersensitivity reactions, syk kinasehelps regulate the pathways involved in immediate IgE-mediated, Type Ihypersensitivity reactions. Certain compounds that affect the sykpathway may or may not also affect the JAK pathways.

Suitable syk inhibitory compounds are described, for example, in Ser.No. 10/355,543 filed Jan. 31, 2003 (publication no. 2004/0029902); WO03/063794; Ser. No. 10/631,029 filed Jul. 29, 2003; WO 2004/014382; Ser.No. 10/903,263 filed Jul. 30, 2004; PCT/US2004/24716 filed Jul. 30, 2004(WO005/016893); Ser. No. 10/903,870 filed Jul. 30, 2004;PCT/US2004/24920 filed Jul. 30, 2004; Ser. No. 60/630,808 filed Nov. 24,2004; Ser. No. 60/645,424 filed Jan. 19, 2005; and Ser. No. 60/654,620,filed Feb. 18, 2005, the disclosures of which are incorporated herein byreference. The described herein and syk inhibitory compounds could beused alone or in combination with one or more conventional transplantrejection treatments, as described above.

In a specific embodiment, the compounds can be used to treat or preventthese diseases in patients that are either initially non-responsive(resistant) to or that become non-responsive to treatment with a sykinhibitory compound or one of the other current treatments for theparticular disease. The compounds could also be used in combination withsyk inhibitory compounds in patients that are syk-compound resistant ornon-responsive. Suitable syk-inhibitory compounds with which thecompounds can be administered are provided infra.

In another embodiment, this invention provides a method of treating aT-cell mediated autoimmune disease, comprising administering to apatient suffering from such an autoimmune disease an amount of acompound effective to treat the autoimmune disease wherein the compoundis selected from the compounds of the invention, as described herein,and the compound is administered in combination with or adjunctively toa compound that inhibits syk kinase with an IC₅₀ in the range of atleast 10 μM.

In another embodiment, this invention provides a method of treating orpreventing allograft transplant rejection in a transplant recipient,comprising administering to the transplant recipient an amount of acompound effective to treat or prevent the rejection wherein thecompound is selected from the compounds of the invention, as describedherein. In a further embodiment, the compound is administered to atissue or an organ prior to transplanting the tissue or organ in thetransplant recipient.

In another embodiment, this invention provides a method of treating orpreventing allograft transplant rejection in a transplant recipient, inwhich the rejection is acute rejection, comprising administering to thetransplant recipient an amount of a compound effective to treat orprevent the rejection, wherein the compound is selected from thecompounds of the invention.

In another embodiment, this invention provides a method of treating orpreventing allograft transplant rejection in a transplant recipient, inwhich the rejection is chronic rejection, comprising administering tothe transplant recipient an amount of a compound effective to treat orprevent the rejection, wherein the compound is selected from thecompounds of the invention.

In another embodiment, this invention provides a method of treating orpreventing allograft transplant rejection in a transplant recipient, inwhich the rejection is mediated by HVGR or GVHR, comprisingadministering to the transplant recipient an amount of a compoundeffective to treat or prevent the rejection, wherein the compound isselected from the compounds of this invention, as described herein.

In another embodiment, this invention provides a method of treating orpreventing allograft transplant rejection in a transplant recipient, inwhich the allograft transplant is selected from a kidney, a heart, aliver, and a lung, comprising administering to the transplant recipientan amount of a compound effective to treat or prevent the rejection,wherein the compound is selected from the compounds of this invention,as described herein.

In another embodiment, this invention provides a method of treating orpreventing allograft transplant rejection in a transplant recipient, inwhich the allograft transplant is selected from a kidney, a heart, aliver, and a lung, comprising administering to the transplant recipientan amount of a compound effective to treat or prevent the rejectionwherein the compound is selected from the compounds of the invention, asdescribed herein, in which the compound is administered in combinationwith or adjunctively to another immunosuppressant.

In another embodiment, this invention provides a method of treating orpreventing allograft transplant rejection in a transplant recipient, inwhich the allograft transplant is selected from a kidney, a heart, aliver, and a lung, comprising administering to the transplant recipientan amount of a compound effective to treat or prevent the rejection,wherein the compound is selected from the compounds of the invention, asdescribed herein, in which the compound is administered in combinationwith or adjunctively to another immunosuppressant, in which theimmunosuppressant is selected from cyclosporine, tacrolimus, sirolimus,an inhibitor of IMPDH, mycophenolate, mycophanolate mofetil, ananti-T-Cell antibody, and OKT3.

The compounds described herein are cytokine moderators of IL-4signaling. As a consequence, the compounds could slow the response ofType I hypersensitivity reactions. Thus, in a specific embodiment, thecompounds could be used to treat such reactions and, therefore, thediseases associated with, mediated by, or caused by suchhypersensitivity reactions (for example, allergies), prophylactically.For example, an allergy sufferer could take one or more of the JAKselective compounds described herein prior to expected exposure toallergens to delay the onset or progress of, or eliminate altogether, anallergic response.

When used to treat or prevent such diseases, the compounds can beadministered singly, as mixtures of one or more compounds, or in mixtureor combination with other agents useful for treating such diseasesand/or the symptoms associated with such diseases. The compounds mayalso be administered in mixture or in combination with agents useful totreat other disorders or maladies, such as steroids, membranestabilizers, 5-lipoxygenase (5LO) inhibitors, leukotriene synthesis andreceptor inhibitors, inhibitors of IgE isotype switching or IgEsynthesis, IgG isotype switching or IgG synthesis, beta.-agonists,tryptase inhibitors, aspirin, cyclooxygenase (COX) inhibitors,methotrexate, anti-TNF drugs, anti CD20 antibody, PD4 inhibitors, p38inhibitors, PDE4 inhibitors, and antihistamines, to name a few. Thecompounds can be administered per se in the form of prodrugs or aspharmaceutical compositions, comprising an active compound or prodrug.

In another embodiment, this invention provides a method of treating orpreventing a Type IV hypersensitivity reaction, comprising administeringto a subject an amount of a compound effective to treat or prevent thehypersensitivity reaction, wherein the compound is selected from thecompounds of this invention, as described herein.

In another embodiment, this invention provides a method of treating orpreventing a Type IV hypersensitivity reaction, which is practicalprophylactically, comprising administering to a subject an amount of acompound effective to treat or prevent the hypersensitivity reaction,wherein the compound is selected from the compounds of this invention,as described herein, and is administered prior to exposure to anallergen.

In another embodiment, this invention provides a method of inhibiting asignal transduction cascade in which JAK3 kinase plays a role,comprising contacting a cell expressing a receptor involved in such asignaling cascade with a compound wherein the compound is selected fromthe compounds of this invention, as described herein.

In another embodiment, this invention provides a method of treating orpreventing a JAK kinase-mediated disease, comprising administering to asubject an amount of compound effective to treat or prevent the JAKkinase-mediated disease, wherein the compound is selected from thecompounds of this invention, as described herein.

In another embodiment, this invention provides a method of treating orpreventing a JAK kinase-mediated disease, in which the JAK-mediateddisease is HVGR or GVHR, comprising administering to a subject an amountof compound effective to treat or prevent the JAK kinase-mediateddisease, wherein the compound is selected from the compounds of theinvention, as described herein.

In another embodiment, this invention provides a method of treating orpreventing a JAK kinase-mediated disease, in which the JAK-mediateddisease is acute allograft rejection, comprising administering to asubject an amount of compound effective to treat or prevent the JAKkinase-mediated disease, wherein the compound is selected from thecompounds of the invention, as described herein.

In another embodiment, this invention provides a method of treating orpreventing a syk and/or JAK kinase-mediated disease, in which theJAK-mediated disease is chronic allograft rejection, comprisingadministering to a subject an amount of compound effective to treat orprevent the JAK kinase-mediated disease, wherein the compound isselected from the compounds of the invention, as described herein.

Active compounds of the invention typically inhibit the syk and/orJAK/Stat pathway. The activity of a specified compound as an inhibitorof a syk and/or JAK kinase can be assessed in vitro or in vivo. In someembodiments, the activity of a specified compound can be tested in acellular assay.

“Cell proliferative disorder” refers to a disorder characterized byabnormal proliferation of cells. A proliferative disorder does not implyany limitation with respect to the rate of cell growth, but merelyindicates loss of normal controls that affect growth and cell division.Thus, in some embodiments, cells of a proliferative disorder can havethe same cell division rates as normal cells but do not respond tosignals that limit such growth. Within the ambit of “cell proliferativedisorder” is neoplasm or tumor, which is an abnormal growth of tissue.Cancer refers to any of various malignant neoplasms characterized by theproliferation of cells that have the capability to invade surroundingtissue and/or metastasize to new colonization sites.

Generally, cell proliferative disorders treatable with the compoundsdisclosed herein relate to any disorder characterized by aberrant cellproliferation. These include various tumors and cancers, benign ormalignant, metastatic or non-metastatic. Specific properties of cancers,such as tissue invasiveness or metastasis, can be targeted using themethods described herein. Cell proliferative disorders include a varietyof cancers, including, among others, ovarian cancer, renal cancer,gastrointestinal cancer, kidney cancer, bladder cancer, pancreaticcancer, lung squamous carcinoma, and adenocarcinoma.

In some embodiments, the cell proliferative disorder treated is ahematopoietic neoplasm, which is aberrant growth of cells of thehematopoietic system. Hematopoietic malignancies can have its origins inpluripotent stem cells, multipotent progenitor cells, oligopotentcommitted progenitor cells, precursor cells, and terminallydifferentiated cells involved in hematopoiesis. Some hematologicalmalignancies are believed to arise from hematopoietic stem cells, whichhave the ability for self renewal. For instance, cells capable ofdeveloping specific subtypes of acute myeloid leukemia (AML) (Cynthia K.Hahn, Kenneth N. Ross, Rose M. Kakoza, Steven Karr, Jinyan Du, Shao-EOng, Todd R. Golub, Kimberly Stegmaier, Syk is a new target for AMLdifferentiation, Blood, 2007, 110, Abstract 209) upon transplantationdisplay the cell surface markers of hematopoietic stem cells,implicating hematopoietic stem cells as the source of leukemic cells.Blast cells that do not have a cell marker characteristic ofhematopoietic stem cells appear to be incapable of establishing tumorsupon transplantation (Blaire et al., 1997, Blood 89:3104-3112). The stemcell origin of certain hematological malignancies also finds support inthe observation that specific chromosomal abnormalities associated withparticular types of leukemia can be found in normal cells ofhematopoietic lineage as well as leukemic blast cells. For instance, thereciprocal translocation t(9q34; 22q11) associated with approximately95% of chronic myelogenous leukemia appears to be present in cells ofthe myeloid, erythroid, and lymphoid lineage, suggesting that thechromosomal aberration originates in hematopoietic stem cells. Asubgroup of cells in certain types of CML displays the cell markerphenotype of hematopoietic stem cells.

Although hematopoietic neoplasms often originate from stem cells,committed progenitor cells or more terminally differentiated cells of adevelopmental lineage can also be the source of some leukemias. Forexample, forced expression of the fusion protein Bcr/Abl (associatedwith chronic myelogenous leukemia) in common myeloid progenitor orgranulocyte/macrophage progenitor cells produces a leukemic-likecondition. Moreover, some chromosomal aberrations associated withsubtypes of leukemia are not found in the cell population with a markerphenotype of hematopoietic stem cells, but are found in a cellpopulation displaying markers of a more differentiated state of thehematopoietic pathway (Turhan et al., 1995, Blood 85:2154-2161). Thus,while committed progenitor cells and other differentiated cells may haveonly a limited potential for cell division, leukemic cells may haveacquired the ability to grow unregulated, in some instances mimickingthe self-renewal characteristics of hematopoietic stem cells (Passegueet al., Proc. Natl. Acad. Sci. USA, 2003, 100:11842-9).

In some embodiments, the hematopoietic neoplasm treated is a lymphoidneoplasm, where the abnormal cells are derived from and/or display thecharacteristic phenotype of cells of the lymphoid lineage. Lymphoidneoplasms can be subdivided into B-cell neoplasms, T and NK-cellneoplasms, and Hodgkin's lymphoma. B-cell neoplasms can be furthersubdivided into precursor B-cell neoplasm and mature/peripheral B-cellneoplasm. Exemplary B-cell neoplasms are precursor B-lymphoblasticleukemia/lymphoma (precursor B-cell acute lymphoblastic leukemia) whileexemplary mature/peripheral B-cell neoplasms are B-cell chroniclymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocyticleukemia, lymphoplasmacytic lymphoma, splenic marginal zone B-celllymphoma, hairy cell leukemia, plasma cell myeloma/plasmacytoma,extranodal marginal zone B-cell lymphoma of MALT type, nodal marginalzone B-cell lymphoma, follicular lymphoma, mantle-cell lymphoma, diffuselarge B-cell lymphoma, mediastinal large B-cell lymphoma, primaryeffusion lymphoma, and Burkitt's lymphoma/Burkitt cell leukemia. T-celland Nk-cell neoplasms are further subdivided into precursor T-cellneoplasm and mature (peripheral) T-cell neoplasms. Exemplary precursorT-cell neoplasm is precursor T-lymphoblastic lymphoma/leukemia(precursor T-cell acute lymphoblastic leukemia) while exemplary mature(peripheral) T-cell neoplasms are T-cell prolymphocytic leukemia T-cellgranular lymphocytic leukemia, aggressive NK-cell leukemia, adult T-celllymphoma/leukemia (HTLV-1), extranodal NK/T-cell lymphoma, nasal type,enteropathy-type T-cell lymphoma, hepatosplenic gamma-delta T-celllymphoma, subcutaneous panniculitis-like T-cell lymphoma, Mycosisfungoides/Sezary syndrome, Anaplastic large-cell lymphoma, T/null cell,primary cutaneous type, Peripheral T-cell lymphoma, not otherwisecharacterized, Angioimmunoblastic T-cell lymphoma, Anaplastic large-celllymphoma, T/null cell, primary systemic type. The third member oflymphoid neoplasms is Hodgkin's lymphoma, also referred to as Hodgkin'sdisease. Exemplary diagnosis of this class that can be treated with thecompounds include, among others, nodular lymphocyte-predominantHodgkin's lymphoma, and various classical forms of Hodgkin's disease,exemplary members of which are Nodular sclerosis Hodgkin's lymphoma(grades 1 and 2), Lymphocyte-rich classical Hodgkin's lymphoma, Mixedcellularity Hodgkin's lymphoma, and Lymphocyte depletion Hodgkin'slymphoma. In various embodiments, any of the lymphoid neoplasms that areassociated with aberrant JAK activity can be treated with the syk and/orJAK inhibitory compounds.

In some embodiments, the hematopoietic neoplasm treated is a myeloidneoplasm. This group comprises a large class of cell proliferativedisorders involving or displaying the characteristic phenotype of thecells of the myeloid lineage. Myeloid neoplasms can be subdivided intomyeloproliferative diseases, myelodysplastic/myeloproliferativediseases, myelodysplastic syndromes, and acute myeloid leukemias.Exemplary myeloproliferative diseases are chronic myelogenous leukemia(e.g., Philadelphia chromosome positive (t(9; 22)(qq34; q11)), chronicneutrophilic leukemia, chronic eosinophilic leukemia/hypereosinophilicsyndrome, chronic idiopathic myelofibrosis, polycythemia vera, andessential thrombocythemia. Exemplary myelodysplastic/myeloproliferativediseases are chronic myelomonocytic leukemia, atypical chronicmyelogenous leukemia, and juvenile myelomonocytic leukemia. Exemplarymyelodysplastic syndromes are refractory anemia, with ringedsideroblasts and without ringed sideroblasts, refractory cytopenia(myelodysplastic syndrome) with multilineage dysplasia, refractoryanemia (myelodysplastic syndrome) with excess blasts, 5q-syndrome, andmyelodysplastic syndrome. In various embodiments, any of the myeloidneoplasms that are associated with aberrant syk and/or JAK activity canbe treated with the syk and/or JAK inhibitory compounds.

In some embodiments, the compounds can be used to treat Acute myeloidleukemias (AML), which represent a large class of myeloid neoplasmshaving its own subdivision of disorders. These subdivisions include,among others, AMLs with recurrent cytogenetic translocations, AML withmultilineage dysplasia, and other AML not otherwise categorized.Exemplary AMLs with recurrent cytogenetic translocations include, amongothers, AML with t(8; 21)(q22; q22), AML1(CBF-alpha)/ETO, Acutepromyelocytic leukemia (AML with t(15; 17)(q22; q11-12) and variants,PML/RAR-alpha), AML with abnormal bone marrow eosinophils(inv(16)(p13q22) or t(16; 16)(p13; q11), CBFb/MYH11X), and AML with11q23 (MLL) abnormalities. Exemplary AML with multilineage dysplasia arethose that are associated with or without prior myelodysplasticsyndrome. Other acute myeloid leukemias not classified within anydefinable group include, AML minimally differentiated, AML withoutmaturation, AML with maturation, Acute myelomonocytic leukemia, Acutemonocytic leukemia, Acute erythroid leukemia, Acute megakaryocyticleukemia, Acute basophilic leukemia, and Acute panmyelosis withmyelofibrosis.

“Treating” within the context of the invention means an alleviation ofsymptoms associated with a disorder or disease, or halt of furtherprogression or worsening of those symptoms, or prevention or prophylaxisof the disease or disorder.

The term “mammal” includes organisms which express syk and/or JAK.Examples of mammals include mice, rats, cows, sheep, pigs, goats,horses, bears, monkeys, dogs, cats and, preferably, humans. Transgenicorganisms which express syk and/or JAK are also included in thisdefinition.

The inventive methods comprise administering an effective amount of acompound or composition described herein to a mammal or non-humananimal. As used herein, “effective amount” of a compound or compositionof the invention includes those amounts that antagonize or inhibit sykand/or JAK. An amount which antagonizes or inhibits syk and/or JAK isdetectable, for example, by any assay capable of determining syk and/orJAK activity, including the one described below as an illustrativetesting method. Effective amounts may also include those amounts whichalleviate symptoms of a syk and/or JAK associated disorder treatable byinhibiting syk and/or JAK. Accordingly, “antagonists of syk” or“antagonists of JAK” include compounds which interact with the syk orJAK, respectively, and modulate, e.g., inhibit or decrease, the abilityof a second compound, e.g., another syk or JAK ligand, to interact withthe syk or JAK, respectively. The syk or JAK binding compounds arepreferably antagonists of syk or JAK, respectively. The language “sykbinding compound” and “JAK-binding compound” (e.g., exhibits bindingaffinity to the receptor) includes those compounds which interact withsyk or JAK resulting in modulation of the activity of syk or JAK,respectively. syk and/or JAK binding compounds may be identified usingan in vitro (e.g., cell and non-cell based) or in vivo method. Adescription of in vitro methods are provided below.

The amount of compound present in the methods and compositions describedherein should be sufficient to cause a detectable decrease in theseverity of the disorder, as measured by any of the assays described inthe examples. The amount of syk and/or JAK modulator needed will dependon the effectiveness of the modulator for the given cell type and thelength of time required to treat the disorder. In certain embodiments,the compositions of this invention may further comprise anothertherapeutic agent. When a second agent is used, the second agent may beadministered either as a separate dosage form or as part of a singledosage form with the compounds or compositions of this invention. Whileone or more of the inventive compounds can be used in an application ofmonotherapy to treat a disorder, disease or symptom, they also may beused in combination therapy, in which the use of an inventive compoundor composition (therapeutic agent) is combined with the use of one ormore other therapeutic agents for treating the same and/or other typesof disorders, symptoms and diseases. Combination therapy includesadministration of the two or more therapeutic agents concurrently orsequentially. The agents may be administered in any order.Alternatively, the multiple therapeutic agents can be combined into asingle composition that can be administered to the patient. Forinstance, a single pharmaceutical composition could comprise thecompound or pharmaceutically acceptable salt, ester or prodrug thereofaccording to the formula I, another therapeutic agent (e.g.,methotrexate) or a pharmaceutically acceptable salt, ester or prodrugthereof, and a pharmaceutically acceptable excipient or carrier.

The invention comprises a compound having the formula I, a method formaking an inventive compound, a method for making a pharmaceuticalcomposition from at least one inventive compound and at least onepharmaceutically acceptable carrier or excipient, and a method of usingone or more inventive compounds to treat a variety of disorders,symptoms and diseases (e.g., inflammatory, autoimmune, neurological,neurodegenerative, oncology and cardiovascular), such as RA,osteoarthritis, irritable bowel disease IBD, asthma, chronic obstructivepulmonary disease COPD and MS. The inventive compounds and theirpharmaceutically acceptable salts and/or neutral compositions may beformulated together with a pharmaceutically acceptable excipient orcarrier and the resulting composition may be administered in vivo tomammals, such as men, women and animals, to treat a variety ofdisorders, symptoms and diseases. Furthermore, the inventive compoundscan be used to prepare a medicament that is useful for treating avariety of disorders, symptoms and diseases.

All of the compounds of the present invention are either potentinhibitors of syk and/or JAK kinases, exhibiting IC₅₀s in the respectiveassay in the range of less than 5 μM, with most being in the nanomolar,and several in the sub-nanomolar, range. In some embodiments, thecompounds of the present invention may be “dual” syk/JAK inhibitors inthat they inhibit both syk and JAK kinase to some degree. In otherembodiments, the compounds of the present invention may selectivelyinhibit syk kinase, but not appreciably inhibit one or more JAK kinases.In other embodiments, the compounds of the present invention mayselectively inhibit JAK kinase, but not appreciably inhibit one or moresyk kinases.

f. Kits

Still another aspect of this invention is to provide a kit comprisingseparate containers in a single package, wherein the inventivepharmaceutical compounds, compositions and/or salts thereof are used incombination with pharmaceutically acceptable carriers to treat states,disorders, symptoms and diseases where syk and/or JAK plays a role.

EXAMPLES

The following examples are offered to illustrate, but not to limit, theclaimed invention.

The starting materials and reagents used in preparing these compoundsgenerally are either available from commercial suppliers, such asAldrich Chemical Co., or are prepared by methods known to those skilledin the art following procedures set forth in references such as Fieserand Fieser's Reagents for Organic Synthesis; Wiley & Sons: New York,1967-2004, Volumes 1-22; Rodd's Chemistry of Carbon Compounds, ElsevierScience Publishers, 1989, Volumes 1-5 and Supplementals; and OrganicReactions, Wiley & Sons: New York, 2005, Volumes 1-65.

The starting materials and the intermediates of the synthetic reactionschemes can be isolated and purified if desired using conventionaltechniques, including but not limited to, filtration, distillation,crystallization, chromatography, and the like. Such materials can becharacterized using conventional means, including physical constants andspectral data.

Unless specified to the contrary, the reactions described hereinpreferably are conducted under an inert atmosphere at atmosphericpressure at a reaction temperature range of from about −78° C. to about150° C., more preferably from about 0° C. to about 125° C., and mostpreferably and conveniently at about room (or ambient) temperature,e.g., about 20° C. to about 75° C.

Referring to the examples that follow, compounds of the presentinvention were synthesized using the methods described herein, or othermethods, which are well known in the art.

The compounds and/or intermediates may be characterized by highperformance liquid chromatography (HPLC) using a Waters Alliancechromatography system with a 2695 Separation Module (Milford, Mass.).The analytical columns may be C-18 SpeedROD RP-18E Columns from MerckKGaA (Darmstadt, Germany). Alternately, characterization may beperformed using a Waters Unity (HPLC) system with Waters Acquity HPLCBEH C-18 2.1 mm×15 mm columns. A gradient elution may be used, typicallystarting with 5% acetonitrile/95% water and progressing to 95%acetonitrile over a period of 5 minutes for the Alliance system and 1minute for the Acquity system. All solvents may contain 0.1%trifluoroacetic acid (TFA). Compounds may be detected by ultravioletlight (UV) absorption at either 220 nm or 254 nm. HPLC solvents may befrom EMD Chemicals, Inc. (Gibbstown, N.J.). In some instances, puritymay be assessed by thin layer chromatography (TLC) using glass backedsilica gel plates, such as, for example, EMD Silica Gel 60 2.5 cm×7.5 cmplates. TLC results may be readily detected visually under ultravioletlight, or by employing well known iodine vapor and other variousstaining techniques.

Mass spectrometric analysis may be performed on one of two Agilent 1100series LCMS instruments with acetonitrile/water as the mobile phase. Onesystem may use TFA as the modifier and measure in positive ion mode[reported as MH+, (M+1) or (M+H)+] and the other may use either formicacid or ammonium acetate and measure in both positive [reported as MH⁺,(M+1) or (M+H)⁺] and negative [reported as M−, (M−1) or (M−H)⁻] ionmodes.

Nuclear magnetic resonance (NMR) analysis may be performed on some ofthe compounds with a Varian 400 MHz NMR (Palo Alto, Calif.). Thespectral reference may be either TMS or the known chemical shift of thesolvent.

The purity of some of the invention compounds may be assessed byelemental analysis (Robertson Microlit, Madison, N.J.).

Melting points may be determined on a Laboratory Devices MeI-Tempapparatus (Holliston, Mass.).

Preparative separations may be carried out as needed, using either anSq16x or an Sg100c chromatography system and prepackaged silica gelcolumns all purchased from Teledyne Isco, (Lincoln, Nebr.). Alternately,compounds and intermediates may be purified by flash columnchromatography using silica gel (230-400 mesh) packing material, or byHPLC using a C-18 reversed phase column. Typical solvents employed forthe Isco systems and flash column chromatography may be dichloromethane,methanol, ethyl acetate, hexane, acetone, aqueous hydroxyamine andtriethyl amine. Typical solvents employed for the reverse phase HPLC maybe varying concentrations of acetonitrile and water with 0.1%trifluoroacetic acid.

General Methods

The following synthetic reaction schemes are merely illustrative of somemethods by which the compounds of the present invention can besynthesized, and various modifications to these synthetic reactionschemes can be made and will be suggested to one skilled in the arthaving referred to the disclosure contained in this application.

Example 12-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-(cyclobutylamino)pyrimidine-5-carboxamide1

Step 1: To a stirring solution of carboxylic acid 1.1 (85 g, 540 mmol)in thionyl chloride (425 mL) was added pyridine (8.5 mL, 0.11 mmol),slowly. The reaction was stirred at 75° C. overnight at which time itwas concentrated and dried under vacuum to a light yellow powder whichwas used immediately for the next step.

Step 2: The yellow solid from the previous step was slowly diluted with750 mL of ethanol and refluxed overnight. The next day the reaction wasdetermined to be complete by HPLC and then cooled in an ice bath and thesolid filtered and washed with diethyl ether affording the desired ethylester (1.3) as an off-white powder (91 g, 87% for two steps). MS foundfor C₇H₈N₂O₄ as (M+H)⁺ 185.0.

Step 3: Ester 1.3 (22 g, 120 mmol) was dissolved in phosphorousoxychloride (60 mL, 600 mmol) and the mixture treated withN,N-diethylaniline (27 mL, 167 mmol) and the mixture heated to 105° C.until the reaction was determined to be complete by HPLC. It was thencooled to rt and slowly added to 1 L of crushed ice resulting in theformation of a beige precipitate which was collected by filtration anddried under vacuum affording the desired dichloride (1.4) as a lightyellow powder (22.5 g, 85%). ¹H NMR (DMSO-d₆, 400 MHz): δ 9.13 (s, 1H),4.37 (q, 2H), 1.32 (t, 3H).

Step 4: Dichloropyrimidine 1.4 (5.9 g, 27 mmol) was dissolved inacetonitrile (50 mL) and treated sequentially with diisopropylamine (5.2mL, 30 mmol) followed by cyclobutyl amine (1.9 g, 27 mmol) and stirredat rt until all starting material had been consumed. The reactionmixture was then diluted with water to a total volume of 150 mL and theprecipitate collected by filtration affording the desired product as alight yellow solid (6.02 g, 87%). ¹H NMR (DMSO-d₆, 400 MHz): δ 8.60 (S,1H), 8.48 (d, 1H), 4.52 (m, 1H), 4.29 (q, 2H), 2.30 (m, 2H), 2.04 (m,2H), 1.73 (m, 2H), 1.30 (t, 3H).

Step 5: Ethyl ester 1.5 (6.02 g, 24 mmol) was diluted with 1,4-dioxane(26 mL) followed by aqueous lithium hydroxide (1.0 M, 26 mL, 26 mmol)and stirred at rt until all starting material had been converted to thecarboxylic acid. The reaction was then diluted with water to a totalvolume of 100 mL and acidified to pH=2 with 6 M HCl. The resultingsuspension was then filtered and dried by aspiration giving 3.51 g ofthe carboxylic acid (64%). ¹H NMR (DMSO-d₆, 400 MHz): δ 8.64 (d, 1H),8.74 (s, 1H), 4.50 (m, 1H), 2.31 (m, 2H), 2.03 (m, 2H), 1.72 (m, 2H).

Step 6: Carboxylic acid 1.6 (3.15 g, 15 mmol) was dissolved inN,N-dimethylformamide (70 mL) and treated with HOBt (3.13 g, 23 mmol)and EDC (4.4 g, 23 mmol). After stirring ca. 25 min ammonia (0.5 M in1,4-dioxane, 72 mL, 36 mmol) was added and the reaction stirredovernight. The following morning the reaction was diluted with water toa total volume of 500 mL and the desired product collected by filtrationaffording 3.62 g (74%) of a light-beige solid. ¹H NMR (DMSO-d₆, 400MHz): δ 9.30 (d, 1H), 8.54 (s, 1H), 8.15 (d, 1H), 8.09 (s, 1H), 7.74 (d,1H), 7.64 (m, 2H), 7.51 (t, 1H), 3.77 (m, 1H), 1.79 (m, 2H), 1.74 (m,2H), 1.53 (m, 1H), 1.41 (m, 1H).

Step 7: Benzotriazolyl ether 1.7 (50 mg, 0.17 mmol),1-(4-(4-aminophenyl)piperazin-1-yl)ethanone (prepared from1-acetylpiperazine and 4-fluoronitrobenze in two steps) (45 mg, 0.20mmol) and p-toluenesulfonic acid (30 mg, 0.17 mmol) were diluted with1,4-dioxane (5 mL) and stirred at 120° C. until all starting materialhad been consumed. The reaction was cooled to rt, diluted with water anddirectly purified by preparative HPLC affording the desired product, 1,after lyophilization. MS found for C₂₁H₂₇N₇O₂ as (M+H)⁺ 410.2.

The following compound was prepared using a procedure similar to thatdescribed in Example 1 with reagent A in place of cyclobutylamine inStep 4.

TABLE 6 Ex Reagent No Structure A MW MS Name 2

meta-anisidine 461.526 462.3 2-(4-(4- acetylpiperazin-1-yl)phenylamino)-4- (3- methoxyphenyl- amino)pyrimidine-5- carboxamide 3

cyclopropylamine 395.467 396 2-(4-(4- acetylpiperazin-1-yl)phenylamino)-4- (cyclopropylamino) pyrimidine-5- carboxamide 4

cyclopentylamine 423.521 424 2-(4-(4- acetylpiperazin-1-yl)phenylamino)-4- (cyclopentylamino) pyrimidine-5- carboxamide 5

t-butylamine 411.51 412.3 2-(4-(4- acetylpiperazin-1- yl)phenylamino)-4-(tert- butylamino) pyrimidine-5- carboxamide 6

3-toluidine 445.527 446.1 2-(4-(4- acetylpiperazin-1- yl)phenylamino)-4-(m- tolylamino)pyrimidine- 5-carboxamide 7

isopropylamine 397.483 398.3 2-(4-(4- acetylpiperazin-1-yl)phenylamino)-4- (isopropylamino) pyrimidine-5- carboxamide 8

n-butylamine 411.51 412 2-(4-(4- acetylpiperazin-1- yl)phenylamino)-4-(butylamino) pyrimidine-5- carboxamide 9

2-methoxy ethylamine 413.482 413 2-(4-(4- acetylpiperazin-1-yl)phenylamino)-4- (2- methoxyethylamino) pyrimidine-5- carboxamide 10

3-methoxypropylamine 427.509 428 2-(4-(4- acetylpiperazin-1-yl)phenylamino)-4- (3- methoxypropylamino) pyrimidine-5- carboxamide 11

tetrahydro-2H-pyran-4-amine 439.52 440 2-(4-(4- acetylpiperazin-1-yl)phenylamino)-4- (tetrahydro-2H- pyran-4- ylamino)pyrimidine-5-carboxamide 12

cyclopropylmethylamine 409.494 410.2 2-(4-(4- acetylpiperazin-1-yl)phenylamino)-4- (cyclopropylmethyl- amino)pyrimidine- 5-carboxamide13

propargylamine 393.451 394.2 2-(4-(4- acetylpiperazin-1-yl)phenylamino)-4- (prop-2- ynylamino)pyrimidine- 5-carboxamide 14

methylamine 369.429 370.2 2-(4-(4- acetylpiperazin-1- yl)phenylamino)-4-(methylamino) pyrimidine-5- carboxamide 15

ethylamine 383.456 384.2 2-(4-(4- acetylpiperazin-1- yl)phenylamino)-4-(ethylamino) pyrimidine-5- carboxamide 16

2,2,2-trifluoroethylamine 437.426 438.2 2-(4-(4- acetylpiperazin-1-yl)phenylamino)-4- (2,2,2- trifluoroethylamino) pyrimidine-5-carboxamide 17

(S)-alpha methylbenzylamine 459.554 460 (S)-2-(4-(4- acetylpiperazin-1-yl)phenylamino)-4- (1- phenylethylamino) pyrimidine-5- carboxamide 18

benzylamine 445.527 446 2-(4-(4- acetylpiperazin-1- yl)phenylamino)-4-(benzylamino) pyrimidine-5- carboxamide 19

4-chlorobenzylamine 479.972 480 2-(4-(4- acetylpiperazin-1-yl)phenylamino)-4- (4- chlorobenzylamino) pyrimidine-5- carboxamide 20

(R)-alpha methylbenzylamine 459.554 460 (R)-2-(4-(4- acetylpiperazin-1-yl)phenylamino)-4- (1- phenylethylamino) pyrimidine-5- carboxamide 21

3,4-dichlorobenzylamine 514.417 514 2-(4-(4- acetylpiperazin-1-yl)phenylamino)-4- (3,4- dichlorobenzylamino) pyrimidine-5- carboxamide22

racemic trans-2-hydroxy cyclopropylamine 439.52 440.5 2-(4-(4-acetylpiperazin-1- yl)phenylamino)-4- ((1R)-2- hydroxycyclopentyl-amino)pyrimidine- 5-carboxamide

Example 322,4-bis(4-(4-acetylpiperazin-1-yl)phenylamino)pyrimidine-5-carboxamide

Step 1: Compound 1.4 (Example 1, 1.05 g, 4.8 mmol) was dissolved in 40mL acetonitrile. To it was added sodium thiomethoxide (0.74 g, 10.5mmol). It was stirred for overnight, diluted with ethyl acetate, washedwith brine three times, dried and concentrated in vacuo. It was thenplaced in 20 mL dioxane and 10 mL water. To it was added LiOH hydrate500 mg. The mixture was stirred for 4 hours. To the mixture was added 1NHCl till the pH reaching 3. It was concentrated, extracted with ethylacetate three times. The organic phases were combined, dried andconcentrated in vacuo to afford a white solid. This solid was thendissolved in 30 mL dry DMF. To it were added EDC hydrochloride (1.10 g,5.7 mmol) and HOBt (0.77 g, 5.7 mmol). The mixture was stirred for 30min, and to it was added ammonia (commercial 0.5N solution in dioxane,29 mL, 14.5 mmol). The mixture was stirred for overnight, concentratedin vacuo, diluted with ethyl acetate, washed with brine three times,dried and concentrated in vacuo to give crude compound 30.1. MS foundfor C₇H₉N₃OS₂ as (M+H)⁺ 216.1.

Step 2: Crude compound 30.1 (42 mg, 0.20 mmol) was dissolved in 4 mLNMP. To it was added MCPBA (133 mg, 0.50 mmol). It was stirred at RT for1 hour. To it were then added1-(4-(4-aminophenyl)piperazin-1-yl)ethanone (175 mg, 0.80 mmol) and DIEA(140 μL, 0.80 mmol). The mixture was then stirred in 120° C. bath for 90min. The mixture was then subjected to reverse phase preparative HPLC toisolate the title compound. MS found for C₂₉H₃₅N₉O₃ as (M+H)⁺ 558.2.

Example 334-(1H-indazol-6-ylamino)-2-(4-(4-acetylpiperazin-1-yl)phenylamino)pyrimidine-5-carboxamide

Step 3: Dichloropyrimidine 1.4 (see Example 1; 1.04 g, 4.7 mmol) wasdissolved in NMP (30 mL) and stirred in ice bath. To it were added6-aminoindazole 31.1 (690 mg, 5.2 mmol) and then dropwiseethyldiisopropylamine (DIEA, 1.64 mL, 9.4 mmol). The mixture was stirredfor 40 minutes, and to it was added sodium thiomethoxide (660 mg, 9.4mmol). The mixture was stirred for overnight, diluted with ethylacetate, washed with brine three times, and concentrated in vacuo togive crude compound 31.2 as a light brown solid in quantitative yield.MS found for C₁₅H₁₅N₅O₂S as (M+H)⁺ 330.1.

Step 4: Ethyl ester 31.2 (4.7 mmol) was dissolved in 60 mL THF. To itwere added lithium hydroxide hydrate (236 mg, 5.6 mmol) and 20 mL water.The mixture was stirred for overnight and to it was carefully added 1NHCl solution till pH reaching 2. The mixture was concentrated in vacuoto remove THF. White solid crashed out and was isolated using a Büchnerfunnel. It was washed with water and dried in vacuum oven to givecompound 31.3 (1.14 g, 81%) as a white solid. MS found for C₁₃H₁₁N₅O₂Sas (M+H)⁺ 302.1.

Step 5: Carboxylic acid 31.3 (1.14 g, 3.8 mmol) was dissolved in 30 mLDMF. To it were added EDC hydrochloride (1.09 g, 5.7 mmol) and HOBthydrate (770 mg, 5.7 mmol). The mixture was stirred at RT for 1 hour. Toit was then added ammonia (commercial 0.5N solution in dioxane, 22 mL,11.4 mmol). The mixture was stirred for 2 hours. It was thenconcentrated in vacuo and taken into water and ethyl acetate. Theorganic phase was separated and washed with brine four times. Theorganic phase was then dried over MgSO₄ and concentrated in vacuo toafford compound 31.4 as a light yellow solid (820 mg, 72%). MS found forC₁₃H₁₂N₆OS as (M+H)⁺ 301.1.

Step 6: Compound 31.4 (36 mg, 0.12 mmol) was dissolved in 3 mL NMP. Toit was added MCPBA (65% pure, 48 mg, 0.18 mmol). It was stirred at RTfor 30 minutes. To it then were added1-(4-(4-aminophenyl)piperazin-1-yl)ethanone (53 mg, 0.24 mmol) and pTSA(21 mg, 0.12 mmol). The mixture was stirred for 90 minutes at 120° C.bath. This mixture was then subjected to preparative HPLC to isolate thetitle compound 31. MS found for C₂₄H₂₅N₉O₂ as (M+H)⁺ 472.2.

Example 342-(4-(4-acetylpiperazin-1-yl)-3-chlorophenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 1 with cyclopropylamine in place of cyclobutylaminein Step 4. Synthesis of the chloropiperazinylaniline was accomplished bychlorination of the nitropiperazinyl intermediate, synthesized in amanner similar to that described in Example 36, with NCS, followed byreduction using sulfide platinum. MS found for C₂₀H₂₄N₇O₂Cl as (M+H)⁺430.0. UV: λ=290

Example 352-(4-(4-acetylpiperazin-1-yl)-3-chlorophenylamino)-4-(cyclobutylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 32. MS found for C₂₁H₂₆N₇O₂Cl as (M+H)⁺ 444.0. UV:λ=211, 290

Example 364-(cyclopropylamino)-2-(4-(4-propionylpiperazin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 1 with cyclopropylamine in place ofcyclobutylamine. The piperazinylaniline was synthesized from Bocpiperazine and 4-fluoronitrobenzene, followed by deprotection using HClin dioxane and acylation using propionyl chloride, and finallyhydrogenation using Pd/C. MS found for C₂₁H₂₇N₇O₂ as (M+H)⁺ 410.3. ¹HNMR (CD₃OD, 400 MHz): δ 8.22 (s, 1H), 7.49 (broad s, 2H), 7.06 (d, 2H),3.73 (m, 4H), 3.22 (m, 4H), 2.47 q, 2H), 3.03 (m, 1H), 1.12 (t, 3H),0.90 (m, 2H), 0.70 (m, 2H).

Example 372-(4-(4-(cyclopropanecarbonyl)piperazin-1-yl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 36 with cyclopropylcarbonyl chloride in place ofpropionyl chloride. MS found for C₂₂H₂₇N₇O₂ as (M+H)⁺ 422.4. ¹H NMR(CD₃OD, 400 MHz): δ 8.22 (s, 1H), 7.45 (broad s, 2H), 7.08 (d, 2H), 3.93(m, 4H), 3.73 (m, 4H), 3.02 (m, 1H), 2.01 (m, 1H), 0.88 (m, 6H), 0.69(m, 2H). UV: λ=203, 273.

Example 384-(cyclopropylamino)-2-(4-(4-(2-methoxyacetyl)piperazin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 36 with methoxyacetyl chloride in place ofpropionyl chloride. MS found for C₂₁H₂₇N₇O₃ as (M+H)⁺ 426.2. ¹H NMR(CD₃OD, 400 MHz): δ 8.38 (s, 1H), 7.55 (broad s, 2H), 7.08 (d, 2H), 4.21(s, 2H), 3.74 (m, 4H), 3.64 (m, 4H), 3.40 (s, 3H), 3.06 (m, 1H), 0.94(m, 2H), 0.71 (m, 2H).

Example 392-(4-(4-acetyl-2-oxopiperazin-1-yl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 1 with cyclopropylamine in place of cyclobutylaminein Step 4. The oxopiperazinyl aniline was synthesized from4-nitroiodobenzene and 4-Boc-2-oxopiperidine using copperiodide/dimethylethylenediamine catalyzed conditions. The Boc group wasthen removed using HCl in dioxane, the resulting amine was acylatedusing acetyl chloride, and finally the nitro group was reduced usinghydrogen and Pd/C. MS found for C₂₀H₂₃N₇O₃ as (M+H)⁺ 410.2. UV: λ=275.

Example 402-(4-(4-acetylpiperazin-1-yl)-3-fluorophenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 1 with cyclopropylamine in place of cyclobutylaminein Step 4. The aniline was synthesized from the corresponding carboxylazide by heating in water/DMF. The azide was ultimately synthesized from3,4-difluorobenzoic acid. MS found for C₂₀H₂₄N₇O₂F as (M+H)⁺ 414.2. UV:λ=293.

Example 412-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-((1s,4s)-4-aminocyclohexylamino)pyrimidine-5-carboxamide

To a mixture of trans-4-aminocyclohexanol (2.07 g, 13.6 mmol) and NaHCO₃(3.50 g, 41.7 mmol) in H₂O (20 mL) at room temperature, a solution ofbenzyl chloroformate (1.92 mL, 13.6 mmol) in dioxane (15 mL) was added.The mixture was stirred at room temperature for 20 h. The whiteprecipitate was collected as benzyl (1R,4R)-4-hydroxycyclohexylcarbamate(3.37 g).

To a suspension of benzyl (1R,4R)-4-hydroxycyclohexylcarbamate (1.14 g,4.58 mmol) and triethylamine (1.30 mL, 9.34 mmol) in CH₂Cl₂ (15 mL) atroom temperature, methanesulfonyl chloride (0.425 mL, 5.49 mmol) wasadded. The mixture was stirred at room temperature for 20 h. Moremethanesulfonyl chloride (0.425 mL, 5.49 mmol) and triethylamine (1.00mL) were added. Stirring was continued for 48 h. The reaction solutionwas washed with 5% NaHCO₃, then with 1 N HCl. The organic phase wasseparated, dried over Na2SO4, concentrated in vacuo to give(1R,4R)-4-(benzyloxycarbonyl)cyclohexyl methanesulfonate as a solid(1.13 g).

A mixture of (1R,4R)-4-(benzyloxycarbonyl)cyclohexyl methanesulfonate(1.13 g, 3.46 mmol) and NaN₃ (0.674 g, 10.4 mmol) in DMF (10 mL) wasstirred at 100 C for 20 h. Water and EtOAc were added. The organic phasewas separated, washed with water, dried over Na₂SO₄, concentrated invacuo to give benzyl (1s,4s)-4-azidocyclohexylcarbamate (0.819 g).

To a solution of benzyl (1s,4s)-4-azidocyclohexylcarbamate (0.410 g,1.50 mmol) in THF (8 mL) and H₂O (0.100 mL, 5.56 mmol) at roomtemperature, Ph₃P (0.590 g, 2.25 mmol) was added. The solution wasstirred at 70 C for 20 h. EtOAc and 1N HCl were added. The aqueous phasewas separated, washed with EtOAc. It was then basified with 5N NaOH topH 12. The free amine product was extracted with EtOAc. The EtOAcsolution was dried over Na₂SO₄, and concentrated in vacuo to give benzyl(1s,4s)-4-aminocyclohexylcarbamate (0.270 g).

A mixture of ethyl 2,4-dichloropyrimidine-5-carboxylate (0.241 g, 1.09mmol), benzyl (1s,4s)-4-aminocyclohexylcarbamate (0.270 g, 1.09 mmol)and triethylamine (0.300 mL, 2.16 mmol) in CH₃CN (10 mL) was stirred atroom temperature for 20 h. Water and EtOAc were added. The organic phasewas separated, washed with 1N HCl, then with 5% NaHCO3, dried overNa2SO4, concentrated in vacuo to give ethyl4-((1s,4s)-4-(benzyloxycarbonyl)cyclohexylamino)-2-chloropyrimidine-5-carboxylate(0.458 g).

To a solution of ethyl4-((1s,4s)-4-(benzyloxycarbonyl)cyclohexylamino)-2-chloropyrimidine-5-carboxylate(0.458 g, 1.06 mmol) in THF (5 mL), aq. 1N LiOH (1.16 mL, 1.16 mmol) wasadded. After being stirred for 3 h, water (10 mL) was added. Thesolution was acidified with 1N HCl (2 mL) to pH 1-2. The product wasextracted with EtOAc. The EtOAc solution was washed with brine, driedover Na₂SO₄, concentrated in vacuo to give4-((1s,4s)-4-(benzyloxycarbonyl)cyclohexylamino)-2-chloropyrimidine-5-carboxylicacid as a solid (0.409 g).

To a solution of4-((1s,4s)-4-(benzyloxycarbonyl)cyclohexylamino)-2-chloropyrimidine-5-carboxylicacid (0.409 g, 1.01 mmol) and HOBt (0.232 g, 1.52 mmol) in DMF (5 mL),EDC (0.291 g, 1.52 mmol) was added. After being stirred for 2 h, NH3(0.5 M in dioxane, 6.0 mL, 3.00 mmol) was added. The mixture was stirredfor 20 h. Water and EtOAc were added. The organic phase was separated,washed with 1N HCl, then with 5% NaHCO₃, dried over Na₂SO₄, concentratedin vacuo to give benzyl(1s,4s)-4-(2-(1H-benzo[d][1,2,3]triazol-1-yloxy)-5-carbamoylpyrimidin-4-ylamino)cyclohexylcarbamateas a solid (0.440 g).

A mixture of benzyl(1s,4s)-4-(2-(1H-benzo[d][1,2,3]triazol-1-yloxy)-5-carbamoylpyrimidin-4-ylamino)cyclohexylcarbamate(0.220 g, 0.438 mmol), 1-(4-(4-aminophenyl)piperazin-1-yl)ethanone(0.192 g, 0.877 mmol) and pTsOH monohydrate (0.083 g, 0.437 mmol) indioxane (4 mL) was stirred at 100 C for 3 h. The mixture was thenpurified by HPLC to give benzyl(1s,4s)-4-(2-(4-(4-acetylpiperazin-1-yl)phenylamino)-5-carbamoylpyrimidin-4-ylamino)cyclohexylcarbamate(0.123 g).

A mixture of benzyl(1s,4s)-4-(2-(4-(4-acetylpiperazin-1-yl)phenylamino)-5-carbamoylpyrimidin-4-ylamino)cyclohexylcarbamate(0.123 g, 0.21 mmol) and Pd—C (10%, 40 mg) in MeOH (5 mL, containingthree drops of 6N HCl), was hydrogenated under balloon H₂ for 4 h. Themixture was filtered through celite, and the filtrate was concentratedin vacuo to give the titled compound as a solid. MS 453.45 (M+H).

Example 422-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-(piperidin-4-ylmethylamino)pyrimidine-5-carboxamide

A mixture of ethyl 2,4-dichloropyrimidine-5-carboxylate (0.221 g, 1.00mmol), 1-Boc-4-aminomethylpiperidine hydrochloride (0.251 g, 1.00 mmol)and triethylamine (0.556 mL, 4.00 mmol) in CH₃CN (10 mL) was stirred atroom temperature for 20 h. Water and EtOAc were added. The organic phasewas separated, washed with 1N HCl, then with 5% NaHCO₃, dried overNa₂SO₄, concentrated in vacuo to give ethyl4-((1-(tert-butoxycarbonyl)piperidin-4-yl)methylamino)-2-chloropyrimidine-5-carboxylate(0.390 g).

To a solution of ethyl4-((1-(tert-butoxycarbonyl)piperidin-4-yl)methylamino)-2-chloropyrimidine-5-carboxylate(0.390 g, 0.979 mmol) in THF (5 mL), aq. 1N LiOH (1.10 mL, 1.10 mmol)was added. After being stirred for 20 h, water (10 mL) was added. Thesolution was acidified with 1N HCl (2 mL) to pH 1-2. The product wasextracted with EtOAc. The EtOAc solution was washed with brine, driedover Na2SO4, concentrated in vacuo to give4-((1-(tert-butoxycarbonyl)piperidin-4-yl)methylamino)-2-chloropyrimidine-5-carboxylicacid as a solid (0.353 g).

To a solution of4-((1-(tert-butoxycarbonyl)piperidin-4-yl)methylamino)-2-chloropyrimidine-5-carboxylicacid (0.353 g, 0.953 mmol) and HOBt (0.219 g, 1.43 mmol) in DMF (5 mL),EDC (0.274 g, 1.43 mmol) was added. After being stirred for 1 h, NH₃(0.5 M in dioxane, 5.5 mL, 2.75 mmol) was added. The mixture was stirredfor 20 h. Water and EtOAc were added. The organic phase was separated,washed with 1N HCl, then with 5% NaHCO₃, dried over Na₂SO₄, concentratedin vacuo to give tert-butyl4-((2-(1H-benzo[d][1,2,3]triazol-1-yloxy)-5-carbamoylpyrimidin-4-ylamino)methyl)piperidine-1-carboxylateas a solid (0.410 g).

A mixture of tert-butyl4-((2-(1H-benzo[d][1,2,3]triazol-1-yloxy)-5-carbamoylpyrimidin-4-ylamino)methyl)piperidine-1-carboxylate(0.205 g, 0.438 mmol), 1-(4-(4-aminophenyl)piperazin-1-yl)ethanone(0.192 g, 0.877 mmol) and pTsOH monohydrate (0.166 g, 0.874 mmol) indioxane (4 mL) was stirred at 100 C for 3 h. The mixture was thenpurified by HPLC to give tert-butyl4-((2-(4-(4-acetylpiperazin-1-yl)phenylamino)-5-carbamoylpyrimidin-4-ylamino)methyl)piperidine-1-carboxylate(0.105 g).

A solution of tert-butyl4-((2-(4-(4-acetylpiperazin-1-yl)phenylamino)-5-carbamoylpyrimidin-4-ylamino)methyl)piperidine-1-carboxylate(0.105 g, 0.190 mmol) in TFA (2 mL) was stirred at room temperature for4 h. TFA was removed in vacuo. The residue was purified by HPLC to givethe titled compound (90 mg). MS 453.41 (M+H).

Example 432-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-((1-acetylpiperidin-4-yl)methylamino)pyrimidine-5-carboxamide

To a solution of2-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-(piperidin-4-ylmethylamino)pyrimidine-5-carboxamide(22 mg, 0.049 mmol) and triethylamine (0.040 mL, 0.29 mmol) inacetonitrile (2 mL) at room temperature, acetic anhydride (0.020 mL,0.21 mmol) was added. The solution was stirred at room temperature for 1h. The mixture was then purified by HPLC to give the titled compound (8mg). MS 495.41 (M+H).

Example 442-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-((1-(methylsulfonyl)piperidin-4-yl)methylamino)pyrimidine-5-carboxamide

To a solution of2-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-(piperidin-4-ylmethylamino)pyrimidine-5-carboxamide(22 mg, 0.049 mmol) and triethylamine (0.040 mL, 0.29 mmol) inacetonitrile (2 mL) at room temperature, methanesulfonyl chloride (0.020mL, 0.26 mmol) was added. The solution was stirred at room temperaturefor 1 h. The mixture was then purified by HPLC to give the titledcompound (14 mg). MS 531.36 (M+H).

Example 452-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-((1-methylpiperidin-4-yl)methylamino)pyrimidine-5-carboxamide

To a solution of2-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-(piperidin-4-ylmethylamino)pyrimidine-5-carboxamide(25 mg, 0.055 mmol) and 37% aq. CH₂O (0.020 mL, 0.27 mmol) in MeOH (1mL) and CH3CO2H (0.1 mL) at room temperature, NaBH3CN (23 mg, 0.36 mmol)was added. The solution was stirred for 20 h. The mixture was thenpurified by HPLC to give the titled compound (20 mg). MS 467.46 (M+H).

Example 462-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-((1-carbamoylpiperidin-4-yl)methylamino)pyrimidine-5-carboxamide

A mixture of2-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-(piperidin-4-ylmethylamino)pyrimidine-5-carboxamide(25 mg, 0.055 mmol) and KOCN (20 mg, 0.25 mmol) in acetic acid (2 mL)was stirred at 100 C for 4 h. The mixture was then purified by HPLC togive the titled compound (8 mg). MS 496.45 (M+H).

Example 47 Benzyl3-((2-(4-(4-acetylpiperazin-1-yl)phenylamino)-5-carbamoylpyrimidin-4-ylamino)methyl)piperidine-1-carboxylateExample 482-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-(piperidin-3-ylmethylamino)pyrimidine-5-carboxamide

A mixture of ethyl 2,4-dichloropyrimidine-5-carboxylate (0.221 g, 1.00mmol), 3-aminomethyl-1-N-CBz-piperidine (0.248 g, 1.00 mmol) andtriethylamine (0.300 mL, 2.15 mmol) in CH3CN (10 mL) was stirred at roomtemperature for 20 h. Water and EtOAc were added. The organic phase wasseparated, washed with 1N HCl, then with 5% NaHCO3, dried over Na₂SO₄,concentrated in vacuo to give ethyl4-((1-(benzyloxycarbonyl)piperidin-3-yl)methylamino)-2-chloropyrimidine-5-carboxylate(0.382 g).

To a solution of ethyl4-((1-(benzyloxycarbonyl)piperidin-3-yl)methylamino)-2-chloropyrimidine-5-carboxylate(0.382 g, 0.88 mmol) in THF (5 mL), aq. 1N LiOH (1.00 mL, 1.00 mmol) wasadded. After being stirred for 20 h, water (10 mL) was added. Thesolution was acidified with 1N HCl (2 mL) to pH 1-2. The product wasextracted with EtOAc. The EtOAc solution was washed with brine, driedover Na2SO4, concentrated in vacuo to give4-((1-(benzyloxycarbonyl)piperidin-3-yl)methylamino)-2-chloropyrimidine-5-carboxylicacid (0.350 g).

To a solution of4-((1-(benzyloxycarbonyl)piperidin-3-yl)methylamino)-2-chloropyrimidine-5-carboxylicacid (0.350 g, 0.87 mmol) and HOBt (0.200 g, 1.31 mmol) in DMF (5 mL),EDC (0.250 g, 1.30 mmol) was added. After being stirred for 1 h, NH3(0.5 M in dioxane, 6.0 mL, 3.0 mmol) was added. The mixture was stirredfor 20 h. Water and EtOAc were added. The organic phase was separated,washed with 1N HCl, then with 5% NaHCO3, dried over Na2SO4, concentratedin vacuo to give benzyl3-((2-(1H-benzo[d][1,2,3]triazol-1-yloxy)-5-carbamoylpyrimidin-4-ylamino)methyl)piperidine-1-carboxylate(0.404 g).

A mixture of benzyl3-((2-(1H-benzo[d][1,2,3]triazol-1-yloxy)-5-carbamoylpyrimidin-4-ylamino)methyl)piperidine-1-carboxylate(0.404 g, 0.805 mmol), 1-(4-(4-aminophenyl)piperazin-1-yl)ethanone(0.220 g, 1.00 mmol) and pTsOH monohydrate (0.167 g, 0.879 mmol) indioxane (8 mL) was stirred at 100 C for 3 h. The mixture was thenpurified by HPLC to give benzyl3-((2-(4-(4-acetylpiperazin-1-yl)phenylamino)-5-carbamoylpyrimidin-4-ylamino)methyl)piperidine-1-carboxylate45 (0.270 g).

A mixture of benzyl3-((2-(4-(4-acetylpiperazin-1-yl)phenylamino)-5-carbamoylpyrimidin-4-ylamino)methyl)piperidine-1-carboxylate(90 mg, 0.15 mmol) and Pd—C (10%, 30 mg) in MeOH (10 mL, containing 4drops of 6N HCl) was hydrogenated under balloon H2 for 4 h. The mixturewas filtered through celite. The filtrate was concentrated in vacuo togive the titled compound 46 (56 mg). MS 453.45 (M+H).

Example 49 2-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-((1-carbamo

To a suspension of2-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-(piperidin-3-ylmethylamino)pyrimidine-5-carboxamide(24 mg, 0.053 mmol) in CH3CN (1 mL), a solution of KOCN (27 mg, 0.33mmol) in H2O (1 mL) was added. The suspension became clear. After beingstirred at 70 C for 2 h, the mixture was purified by HPLC to give thetitled compound (14 mg). MS 497 (M+H).

Example 502-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-(5-hydroxypentylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 1 using 5-aminopentanol in place of cyclobutylamine. MS found for C₂₂H₃₁N₇O₃ as (M+H)⁺ 442.0.

Example 51(S)-2-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-(1-hydroxypropan-2-ylamino)pyrimidine-5-carboxamide

Step 1: Conversion of dichloropyrimide 1.4 to thiomethyl 49.1 wasaccomplished using a procedure similar to that described in Synthesisand evaluation of2-{[2-(4-hydroxyphenyl)-ethyl]amino}pyrimidine-5-carboxamide derivativesas novel STAT6 inhibitors. Nagashima, Shinya; Yokota, Masaki; Nakai,Ei-ichi; Kuromitsu, Sadao; Ohga, Keiko; Takeuchi, Makoto; Tsukamoto,Shin-ichi; Ohta, Mitsuaki. Institute for Drug Discovery Research,Astellas Pharm Inc., Yodogawa-ku, Osaka, Japan. Bioorganic & MedicinalChemistry (2007), 15(2), 1044-1055.

Steps 2-4 were accomplished using procedures similar to those describedin Example 1.

Step 5: The above compound was prepared using a procedure similar tothat described in Example 25, Step 2. MS found for C₂₀H₂₇N₇O₃ as (M+H)⁺414.4.

Example 532-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-(cyanomethylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 51. MS found for C₁₉H₂₂N₈O₂ as (M+H)⁺ 395.2. UV:λ=203, 273.

Example 54(R)-2-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-(1-hydroxypropan-2-ylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 51. MS found for C₂₀H₂₇N₇O₃ as (M+H)⁺ 414.3. UV:λ=201, 274

Example 552-(4-(4-acetyl-2-carbamoylpiperazin-1-yl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 1, using cyclopropylamine in place ofcyclobutylamine and Boc protected piperazine carboxylic acid in place ofacetylpiperazine. MS found for C₂₁H₂₆N₈O₃ as (M+H)⁺ 439.4.

Example 56(R)-2-(4-(4-acetyl-3-methylpiperazin-1-yl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 1 with cyclopropylamine in place ofcyclobutylamine. MS found for C₂₁H₂₇N₇O₂ as (M+H)⁺ 410.4.

Example 572-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-(2-amino-2-oxoethylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 51. MS found for C₁₉H₂₄N₈O₃ as (M+H)⁺ 413.4.

Example 582-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-(3-amino-3-oxopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 51. MS found for C₂₀H₂₆N₈O₃ as (M+H)⁺ 427.4. UV:λ=200, 270.

Example 592-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-(4-amino-4-oxobutylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 51. MS found for C₂₁H₂₈N₈O₃ as (M+H)⁺ 441.3.

Example 602-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-(2-cyanoethylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 51. MS found for C₂₀H₂₄N₈O₂ as (M+H)⁺ 409.3. UV:λ=202, 251.

Example 612-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-(1-carbamoylcyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 1. MS found for C₂₁H₂₆N₈O₃ as (M+H)⁺ 439.3.

Example 622-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-(2-morpholinoethylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 51. MS found for C₂₃H₃₂N₈O₃ as (M+H)⁺ 469.4.

Example 632-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-((1R,2R)-2-carbamoylcyclopentylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 51 using an amine prepared using the procedure fromAn Efficient Route to Either Enantiomer oftrans-2-Aminocyclopentanecarboxylic Acid. LePlae, P. R.; Umezawa, N.;Lee, H.-S.; Gellman, S. H. J. Org. Chem.; (Note); 2001; 66(16);5629-5632. MS found for C₂₃H₃₀N₈O₃ as (M+H)⁺ 467.4. UV: λ=202, 258.

Example 642-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-(−2-trans-phenylcyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 51. MS found for C₂₆H₂₉N₇O₂ as (M+H)⁺ 472.3.

Example 652-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-(3-hydroxypropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 51. MS found for C₂₀H₂₇N₇O₃ as (M+H)⁺ 414.3.

Example 662-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-(4-hydroxybutylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 51. MS found for C₂₁H₂₉N₇O₃ as (M+H)⁺ 428.4.

Example 672-(4-(4-acetyl-2-(methylcarbamoyl)piperazin-1-yl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 1 using cyclopropylamine in place ofcyclobutylamine and Boc protected piperazine carboxylic acid in place ofacetylpiperazine. MS found for C₂₂H₂₈N₈O₃ as (M+H)⁺ 453.3.

Example 69(R)-2-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-(2,3-dihydroxypropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 51. MS found for C₂₀H₂₇N₇O₄ as (M+H)⁺ 430.3.

Example 702-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-((1R,3R)-3-carbamoylcyclopentylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example Example 51 using an amine derived Boc protected(1,R, 3R)-3-aminocyclopentane carboxylic acid. MS found for C₂₃H₃₀N₈O₃as (M+H)⁺ 467.4.

Example 71 methyl4-(4-(5-carbamoyl-4-(cyclopropylamino)pyrimidin-2-ylamino)phenyl)piperazine-1-carboxylate

The above compound was prepared using a procedure similar to thatdescribed in Example 1 using cyclopropylamine in place ofcyclobutylamine and a suitable aniline. MS found for C₂₀H₂₅N₇O₃ as(M+H)⁺ 412.3.

Example 722-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-(2-oxo-1,2,3,4-tetrahydroquinolin-6-ylamino)pyrimidine-5-carboxamide

The titled compound was synthesized analogously by using6-amino-3,4-dihydroquinolin-2(1H)-one. MS 501.3 (M+H); UV: λ=207.8,293.8.

Example 734-((1s,4s)-4-aminocyclohexylamino)-2-(4-(piperazin-1-yl)phenylamino)pyrimidine-5-carboxamide

To a solution of2-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-((1s,4s)-4-aminocyclohexylanimo)pyrimidine-5-carboxamide(52 mg, 0.12 mmol) in EtOH (3 mL), aq. 3M KOH (1.0 mL, 3.0 mmol) wasadded. The mixture was stirred at 85 C for 4 h., then at 70 C for 20 h.It was concentrated in vacuo. The residue was acidified with acetic acid(2 mL) before it was purified by HPLC to give the titled compound (25mg). MS 411.43 (M+H); UV: λ=228.5, 285.3.

Example 74(R)-2-(4-(4-acetyl-2-methylpiperazin-1-yl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₂₁H₂₇N₇O₂ as (M+H)⁺ 410.4. UV: λ=209,265.

Example 75(R)-2-(4-(4-acetyl-2-methylpiperazin-1-yl)phenylamino)-4-(2,2,2-trifluoroethylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using trifluoroethylamine in place ofcyclobutylamine. MS found for C₂₀H₂₄F₃N₇O₂ as (M+H)⁺ 452.4. UV: λ=216,276.

Example 76(S)-2-(4-(4-acetyl-2-methylpiperazin-1-yl)phenylamino)-4-(2,2,2-trifluoroethylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using trifluoroethylamine in place ofcyclobutylamine. MS found for C₂₀H₂₄F₃N₇O₂ as (M+H)⁺ 452.4. UV: λ=201,274.

Example 772-(4-(4-carbamoylpiperazin-1-yl)phenylamino)-4-(cyclobutylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 1. MS found for C₂₀H₂₆N₈O₂ as (M+H)⁺ 411.2. UV:λ=204, 262.

Example 784-(cyclobutylamino)-2-(4-(4-(dimethylcarbamoyl)piperazin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 1. MS found for C₂₂H₃₀N₈O₂ as (M+H)⁺ 439.3. UV:λ=206, 263.

Example 792-(6-(4-acetylpiperazin-1-yl)pyridin-3-ylamino)-4-(2,2,2-trifluoroethylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using trifluoroethylamine in place ofcyclobutylamine. MS found for C₁₈H₂₁F₃N₈O₂ as (M+H)⁺ 439.4. ¹H NMR(CD₃OD, 400 MHz): δ 8.52 (s, 1H), 8.49 (s, 1H), 7.98 (dd, 1H), 7.23 (d,1H), 4.52 (q, 2H), 3.74 (m, 6H), 3.64 (m, 2H), 2.15 (s, 3H).

Example 802-(4-(4-acetylpiperazin-1-yl)-2-methylphenylamino)-4-(2,2,2-trifluoroethylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using trifluoroethylamine in place ofcyclobutylamine. MS found for C₂₀H₂₄F₃N₇O₂ as (M+H)⁺ 452.4. UV: λ=204,227.

Example 81(S)-2-(4-(4-(2-methoxyacetyl)-2-methylpiperazin-1-yl)phenylamino)-4-(2,2,2-trifluoroethylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using trifluoroethylamine in place ofcyclobutylamine. MS found for C₂₁H₂₆F₃N₇O₃ as (M+H)⁺ 482.5. UV: λ=202,274.

Example 82 (S)-methyl4-(4-(5-carbamoyl-4-(2,2,2-trifluoroethylamino)pyrimidin-2-ylamino)phenyl)-3-methylpiperazine-1-carboxylate

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using trifluoroethylamine in place ofcyclobutylamine. MS found for C₂₀H₂₄F₃N₇O₃ as (M+H)⁺ 468.5. UV: λ=202,274.

Example 832-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-(1-methyl-1H-indazol-4-ylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 48. MS found for C25H27N9O2 as (M+H)⁺ 486.4. UV:λ=205.8, 299.4.

Example 842-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-(benzo[c]thiadiazol-4-ylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 48. MS found for C23H23N9O2S as (M+H)⁺ 490.4. UV:λ=236.2, 283.4, 305.6.

Example 854-(benzo[c]thiadiazol-4-ylamino)-2-(4-(4-propionylpiperazin-1-yl)phenylamino)-pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 48. MS found for C24H25N9O2S as (M+H)⁺ 504.4. UV:λ=202.8, 236.2, 301.9, 314.9.

Example 864-(1-methyl-1H-indol-4-ylamino)-2-(4-(4-propionylpiperazin-1-yl)phenylamino)pyrimidine-5-carboxamide

The title compound was prepared using the same synthetic schemedemonstrated in Example 33. MS found for C₂₇H₃₀N₈O₂ as (M+H)⁺ 499.4. UV:λ=219.2.

Example 874-(1,2-dimethyl-1H-indol-4-ylamino)-2-(4-(4-propionylpiperazin-1yl)phenylamino)pyrimidine-5-carboxamide

The title compound was prepared using the same synthetic schemedemonstrated in Example 32. MS found for C₂₈H₃₂N₈O₂ as (M+H)⁺ 513.4. UV:λ=208.6.

Example 884-(1-methyl-1H-indol-4-ylamino)-2-(4-(4-propionylpiperazin-1-yl)phenylamino)pyrimidine-5-carboxamide

The title compound was prepared using the same synthetic schemedemonstrated in Example 33. MS found for C₂₇H₃₀N₈O₂ as (M+H)⁺ 499.4. UV:λ=219.2.

Example 894-(1,2-dimethyl-1H-indol-4-ylamino)-2-(4-(4-propionylpiperazin-1-yl)phenylamino)pyrimidine-5-carboxamide

The title compound was prepared using the same synthetic schemedemonstrated in Example 33. MS found for C₂₈H₃₂N₈O₂ as (M+H)⁺ 513.4. UV:λ=208.6.

Example 924-(cyclobutylamino)-2-(2-oxoindolin-5-ylamino)pyrimidine-5-carboxamide

MS found for C₁₅H₁₆N₅OCl as (M+H)⁺ 318.0, 320.0. ¹H NMR (DMSO-d₆, 400MHz): δ 8.32 (s, 1H), 7.56 (d, 2H), 7.41 (d, 2H), 4.51 (m, 1H), 2.43 (m,2H), 2.08 (m, 2H), 1.88 (m, 2H). UV: λ=205, 264.

Example 932-(6-(4-acetylpiperazin-1-yl)pyridin-3-ylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using an intermediate synthesized asdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine,and 1-(4-(5-aminopyridin-2-yl)piperazin-1-yl)ethanone (prepared from1-acetylpiperazine and 2-chloro-5-nitropyridine in two steps). MS foundfor C₁₉H₂₄N₈O₂ as (M+H)⁺ 397.0. ¹H NMR (CD₃OD, 400 MHz): δ 8.62 (s, 1H),8.41 (s, 1H), 8.08 (broad s, 1H), 7.17 (d, 1H) 3.64-3.78 (m, 8H), 2.98(m, 1H), 2.15 (s, 3H), 0.96 (m, 2H), 0.70 (m, 2H).

Example 94(R)-2-(4-(2-methyl-4-(methylsulfonyl)piperazin-1-yl)phenylamino)-4-(2,2,2-trifluoroethylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using trifluoroethylamine in place ofcyclobutylamine. MS found for C₁₉H₂₄F₃N₇O₃S as (M+H)⁺ 488.4.

Example 95(S)-2-(4-(2-methyl-4-(methylsulfonyl)piperazin-1-yl)phenylamino)-4-(2,2,2-trifluoroethylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using trifluoroethylamine in place ofcyclobutylamine. MS found for C₁₉H₂₄F₃N₇O₃S as (M+H)⁺ 488.3. UV: λ=201,275.

Example 964-(cyclopropylamino)-2-(4-(1-(methylsulfonyl)-1,2,3,6-tetrahydropyridin-4-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₂₀H₂₄N₆O₃S as (M+H)⁺ 429.3.

Example 974-(cyclopropylamino)-2-(4-(1-(methylsulfonyl)piperidin-4-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₂₀H₂₆N₆O₃ as (M+H)⁺ 431.3.

Example 984-(cyclopropylamino)-2-(4-(4-(N-methylmethylsulfonamido)piperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1, using cyclopropylamine in place ofcyclobutylamine and an aniline derived from tert-butylpiperidin-4-ylcarbamate and 4-fluoronitrobenzene. MS found forC₂₁H₂₉N₇O₃S as (M+H)⁺ 460.2. UV: λ=202, 272.

Example 994-(cyclopropylamino)-2-(4-(4-(methylsulfonyl)piperazin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1, using cyclopropylamine in place ofcyclobutylamine. MS found for C₁₉H₂₅N₇O₃S as (M+H)⁺ 432.0.

Example 100a4-(cyclopropylamino)-2-(4-(4-(ethylsulfonyl)piperazin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1, using cyclopropylamine in place ofcyclobutylamine. MS found for C₂₀H₂₇N₇O₃S as (M+H)⁺ 446.2. ¹H NMR(CD₃OD, 400 MHz): δ 8.37 (s, 1H), 7.59 (broad s, 2H), 7.09 (d, 2H), 3.45(m, 4H), 3.50 (m, 4H), 3.12 (q, 2H), 3.07 (m, 1H), 1.35 (t, 3H), 0.93(m, 2H), 0.73 (m, 2H). UV: λ=275.

Example 100b4-(cyclobutylamino)-2-(4-(4-(methylsulfonyl)piperazin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 1. MS found for C₂₀H₂₇N₇O₃S as (M+H)⁺ 446.0.

Example 100c2-(2-methyl-4-(4-(methylsulfonyl)piperazin-1-yl)phenylamino)-4-(2,2,2-trifluoroethylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using trifluoroethylamine in place ofcyclobutylamine and an aniline derived from4-fluoro-2-methylnitrobenzene. MS found for C₁₉H₂₄F₃N₇O₃S as (M+H)⁺488.4. UV: λ=227.

Example 101a(S)-2-(4-(4-(ethylsulfonyl)-2-methylpiperazin-1-yl)phenylamino)-4-(2,2,2-trifluoroethylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using trifluoroethylamine in place ofcyclobutylamine. MS found for C₂₀H₂₆F₃N₇O₃S as (M+H)⁺ 502.4. UV: λ=203,274.

Example 102(S)-2-(4-(4-(cyclopropylsulfonyl)-2-methylpiperazin-1-yl)phenylamino)-4-(2,2,2-trifluoroethylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using trifluoroethylamine in place ofcyclobutylamine. MS found for C₂₁H₂₆F₃N₇O₃S as (M+H)⁺ 514.5. UV: λ=202,274.

Example 1034-(cyclobutylamino)-2-(4-sulfamoylphenylamino)pyrimidine-5-carboxamide

The above compound was prepared using 4-aminobenzenesulfonamide using aprocedure similar to that described in Scheme 1. MS found forC₁₅H₁₈N₆O₃S as (M+H)⁺ 363.0.

Example 104 methyl4-(5-carbamoyl-4-(cyclobutylamino)pyrimidin-2-ylamino)phenyl(methyl)carbamate

The above compound was prepared using methyl4-aminophenyl(methyl)carbamate (synthesized from N-methyl 4-nitroanilinein two steps) using a procedure similar to that described in Scheme 1.MS found for C₁₈H₂₂N₆O₃ as (M+H)⁺ 371.0. UV: λ=203, 263.

Example 105 isopropyl4-(5-carbamoyl-4-(cyclobutylamino)pyrimidin-2-ylamino)phenyl(methyl)carbamate

The above compound was prepared using isopropyl4-aminophenyl(methyl)carbamate (synthesized from N-methyl 4-nitroanilinein two steps) using a procedure similar to that described in Scheme 1.MS found for C₂₀H₂₆N₆O₃ as (M+H)⁺ 399.0. UV: λ=277.

Example 1064-(cyclobutylamino)-2-(4-(N,N-dimethylsulfamoyl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using4-amino-N,N-dimethylbenzenesulfonamide (prepared from4-nitrobenzenesulfonic chloride and dimethyl amine followed byreduction) using a procedure similar to that described in Scheme 1. MSfound for C₁₇H₂₂N₆O₃S as (M+H)⁺ 391.0. UV: λ=213, 288.

Example 107 methyl4-(5-carbamoyl-4-(cyclopropylamino)pyrimidin-2-ylamino)phenyl(methyl)carbamate

The above compound was prepared using an intermediate synthesized asdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine,and methyl 4-aminophenyl(methyl)carbamate (synthesized from N-methyl4-nitroaniline in two steps) using a procedure similar to that describedin Scheme 1. MS found for C₁₇H₂₀N₆O₃ as (M+H)⁺ 357.0. UV: λ=204, 273.

Example 108 isopropyl4-(5-carbamoyl-4-(cyclopropylamino)pyrimidin-2-ylamino)phenyl(methyl)carbamate

The above compound was prepared using an intermediate synthesized asdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylaminein Step 4, and isopropyl 4-aminophenyl(methyl)carbamate (synthesizedfrom N-methyl 4-nitroaniline in two steps). MS found for C₁₉H₂₄N₆O₃ as(M+H)⁺ 385.0. UV: λ=201, 276.

Example 1094-(cyclopropylamino)-2-(4-sulfamoylphenylamino)pyrimidine-5-carboxamide

The above compound was prepared using an intermediate synthesized asdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine,and 4-aminobenzenesulfonamide. MS found for C₁₄H₁₆N₆O₃S as (M+H)⁺ 349.0.¹H NMR (DMSO-d₆, 400 MHz): δ 8.38 (s, 1H), 7.93 (d, 2H), 7.73 (d, 2H),4.76 (m, 1H), 2.47 (m, 2H), 2.08 (m, 2H), 1.87 (m, 2H). UV: λ=285.

Example 1104-(cyclopropylamino)-2-(4-(1-methylureido)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1, using cyclopropylamine in place ofcyclobutylamine and an aniline prepared from protectedN′Boc-N-methylphenylene diamine in two steps. MS found for C₁₆H₁₉N₇O₂ as(M+H)⁺ 342.0. UV: λ=205, 272.

Example 1114-(cyclopropylamino)-2-(4-(methylsulfinyl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1, using cyclopropylamine in place ofcyclobutylamine, and using 4-thiomethylaniline followed by oxidationusing mCPBA. MS found for C₁₅H₁₇N₅O₂S as (M+H)⁺ 332.1.

Example 1122-(4-(4-acetylpiperazine-1-carbonyl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1, using cyclopropylamine in place ofcyclobutylamine and an aniline prepared from 4-nitrobenzoyl chloride andN-acetylpiperazine in two steps. MS found for C₂₁H₂₅N₇O₃ as (M+H)⁺424.1.

Example 1132-(4-(1-acetylpiperidin-4-yl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₂₁H₂₆N₆O₂ as (M+H)⁺ 395.3.

Example 114(cyclopropylamino)-2-(4-(4-(pyrrolidine-1-carbonyl)piperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place of cyclobutylamineand an aniline derived from 4-fluoronitrobenzene and ethylisonipecotate. MS found for C₂₄H₃₁N₇O₂ as (M+H)⁺ 450.3. UV: λ=271.

Example 1154-(cyclopropylamino)-2-(4-(4-(piperidine-1-carbonyl)piperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place of cyclobutylamineand an aniline derived from 4-fluoronitrobenzene and ethylisonipecotate. MS found for C₂₅H₃₃N₇O₂ as (M+H)⁺ 464.3. UV: λ=201, 271.

Example 1164-(cyclopropylamino)-2-(4-(4-(morpholine-4-carbonyl)piperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place of cyclobutylamineand an aniline derived from 4-fluoronitrobenzene and ethylisonipecotate. MS found for C₂₄H₃₁N₇O₃ as (M+H)⁺ 466.3. UV: λ=201, 230,285.

Example 1174-(cyclopropylamino)-2-(4-(4-(cyclopropylsulfonyl)piperazin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1. MS found for C₂₁H₂₇N₇O₃S as (M+H)⁺ 458.2. UV:λ=201, 231, 282.

Example 1182-(4-(4-acetyl-1,4-diazepan-1-yl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1. MS found for C₂₁H₂₇N₇O₂ as (M+H)⁺ 410.2. UV:λ=234, 258.

Example 1192-(4-(4-acetamidopiperidin-1-yl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using an aniline derived from 4-Bocaminopiperidine and 4-fluoronitrobenzene which was converted to theacetyl after Boc deprotection, then the nitro group was reduced to theaniline using hydrogenation. MS found for C₂₁H₂₇N₇O₂ as (M+H)⁺ 410.2.

Example 1204-(cyclopropylamino)-2-(4-(2-oxopyridin-1(2H)-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using an aniline derived from 4-iodoaniline and2-hydroxypyridine which were coupled using CuI and a suitable ligand. MSfound for C₂₁H₂₇N₇O₂ as (M+H)⁺ 363.2. UV: λ=201, 274.

Example 1214-(cyclopropylamino)-2-(4-dioxothiomorpholinophenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1. MS found for C₁₈H₂₂N₆O₂S as (M+H)⁺ 403.2.

Example 1224-(cyclopropylamino)-2-(4-(4-(2-methoxyethyl)piperazin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place of cyclobutylamineand an aniline derived from 4-fluoronitrobenzene and1-methoxyethylpiperazine in two steps. MS found for C₂₁H₂₉N₇O₂ as (M+H)⁺412.0.

Example 1234-(cyclopropylamino)-2-(4-(4-(1-methylcyclopropyl)piperazin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₂₂H₂₉N₇O as (M+H)⁺ 408.3.

Example 1244-(cyclopropylamino)-2-(4-(4-(N-methylacetamido)piperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place of cyclobutylamineand an aniline described previously with the methylation of the N-acetylgroup (CH₃I, Cs₂CO₃, DMF) being performed before the nitro reductionstep. MS found for C₂₂H₂₉N₇O₂ as (M+H)⁺ 424.2.

Example 1252-(4-(N-cyclobutylsulfamoyl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamineand an aniline derived from 4-nitrobenzenesulfonyl chloride andcyclobutylamine. MS found for C₁₈H₂₂N₆O₃S as (M+H)⁺ 403.0.

Example 1264-(cyclopropylamino)-2-(4-(N-cyclopropylsulfamoyl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamineand an aniline similar to that described previously. MS found forC₁₇H₂₀N₆O₃S as (M+H)⁺ 389.0.

Example 1274-(cyclopropylamino)-2-(4-(N-(2-methoxyethyl)sulfamoyl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamineand an aniline similar to that described previously. MS found forC₁₇H₂₂N₆O₄S as (M+H)⁺ 407.0.

Example 1284-(cyclopropylamino)-2-(4-(N-(2-hydroxyethyl)sulfamoyl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamineand an aniline similar to that described previously. MS found forC₁₆H₂₀N₆O₄S as (M+H)⁺ 393.0.

Example 1294-(cyclobutylamino)-2-(4-(methylsulfonyl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine.MS found for C₁₆H₁₉N₅O₃S as (M+H)⁺ 362.1. UV: λ=292.

Example 1304-(cyclobutylamino)-2-(4-(piperazin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1. MS found for C₁₉H₂₅N₇O as (M+H)⁺ 368.3. UV:λ=203, 229, 256, 288.

Example 1314-(cyclopropylamino)-2-(4-(4-(pyridin-2-yl)piperazin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine.MS found for C₂₃H₂₆N₈O as (M+H)⁺ 431.4.

Example 1324-(cyclobutylamino)-2-(4-(methylsulfonyl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 1. MS found for C₁₆H₁₉N₅O₃S as (M+H)⁺ 362.1.

Example 1334-(cyclobutylamino)-2-(4-(piperazin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 1. MS found for C₁₉H₂₅N₇O as (M+H)⁺ 368.3.

Example 1344-(cyclopropylamino)-2-(4-(4-(pyridin-2-yl)piperazin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine.MS found for C₂₃H₂₆N₈O as (M+H)⁺ 431.4.

Example 1352-(4-(4-(aminomethyl)piperidin-1-yl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine.MS found for C₂₀H₂₇N₇O as (M+H)⁺ 382.5.

Example 1364-(cyclopropylamino)-2-(4-(N-pyrimidin-2-ylsulfamoyl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine.MS found for C₁₈H₁₈N₈O₃S as (M+H)⁺ 427.0.

Example 1374-(cyclopropylamino)-2-(4-(N-thiazol-2-ylsulfamoyl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine.MS found for C₁₇H₁₇N₇O₃S₂ as (M+H)⁺ 432.0.

Example 1384-(cyclopropylamino)-2-(4-(N-(5-methylisoxazol-3-yl)sulfamoyl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine.MS found for C₁₈H₁₉N₇O₄S as (M+H)⁺ 430.0.

Example 1394-(cyclopropylamino)-2-(4-(N-(2,6-dimethylpyrimidin-4-yl)sulfamoyl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine.MS found for C₂₀H₂₂N₈O₃S as (M+H)⁺ 455.0.

Example 1404-(cyclobutylamino)-2-(4-(1-methylureido)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1. MS found for C₁₇H₂₁N₇O₂ as (M+H)⁺ 356.3. UV:λ=202, 263.

Example 1414-(cyclobutylamino)-2-(4-(trifluoromethoxy)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1. MS found for C₁₆H₁₆N₅O₂F₃ as (M+H)⁺ 368.0.

Example 1432-(4-(2-morpholino-2-oxoethoxy)phenylamino)-4-(m-tolylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1, with meta-toluidine in place of cyclobutylamine.MS found for C₂₄H₂₆N₆O₄ as (M+H)⁺ 463.3. UV: λ=282.

Example 1452-(4-(2-morpholinoethoxy)phenylamino)-4-(m-tolylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1, with meta-toluidine in place of cyclobutylamine.MS found for C24H28N6O3 as (M+H)⁺ 449.3. UV: λ=281.

Example 1474-(cyclopropylamino)-2-(4-(isoxazol-3-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine.MS found for C17H16N6O2 as (M+H)⁺ 337.3. UV: λ=298.5.

Example 148(S)-2-(4-(1-amino-3-methyl-1-oxobutan-2-ylamino)phenylamino)-4-(2,2,2-trifluoroethylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using trufluoroethylamine in place ofcyclobutylamine and an aniline derived from (S)-valine and4-fluoronitrobenzene. MS found for C₁₈H₂₂F₃N₇O₂ as (M+H)⁺ 426.4. UV:λ=225, 294. ¹H NMR (CD₃OD, 400 MHz): δ 8.32 (s, 1H), 7.19 (broad s, 2H),6.76 (d, 2H), 4.35 (broad s, 2H), 3.58 (d, 1H), 2.11 (m, 1H), 1.10 (dd,6H).

Example 149(S)-2-(4-(1-amino-4-methyl-1-oxopentan-2-ylamino)phenylamino)-4-(2,2,2-trifluoroethylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using trifluoroethylamine in place ofcyclobutylamine and an aniline similar to that described previously. MSfound for C₁₉H₂₄F₃N₇O₂ as (M+H)⁺ 440.4. UV: λ=226, 303.

Example 1514-(1-methyl-1H-indol-4-ylamino)-2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenylamino)pyrimidine-5-carboxamide

Step 1: To a solution of ethyl 2,4-dichloropyrimidine-5-carboxylate (328mg, 1.48 mmol) and 1-methyl-1H-indol-4-amine (260 mg, 1.78 mmol) inCH3CN (6 mL) at room temperature, DIEA (0.4 mL, 2.22 mmol) was added.The mixture was stirred at room temperature for 24 h. Water (15 mL) wasadded to induce precipitation. The precipitate was collected, dried onvacuum to give ethyl 2-chloro-4-(1-methyl-1H-indol-4-ylamino)pyrimidine-5-carboxylate as a solid.

Step 2: To a solution of ethyl 2-chloro-4-(1-methyl-1H-indol-4-ylamino)pyrimidine-5-carboxylate (crude from step 1) in THF (4 mL), aq. 1N LiOH(2.25 mL, 2.25 mmol) was added. The mixture was stirred at roomtemperature overnight. Upon acidification of the mixture with 1N HCl,white solids precipitated out, which were collected, and dried on vacuumto give 2-chloro-4-(1-methyl-1H-indol-4-ylamino) pyrimidine-5-carboxylicacid (325 mg). MS 303.3, 305.3 (M+H, Cl pattern)

Step 3: To a solution of2-chloro-4-(1-methyl-1H-indol-4-ylamino)pyrimidine-5-carboxylic acid(325 mg, 1.08 mmol) and HOBt (198 mg, 1.29 mmol) in DMF (4 mL), EDC (248mg, 1.29 mmol) was added. The mixture was stirred at room temperaturefor 1.5 h. Ammonia (0.5 M in dioxane, 8.00 mL, 4.00 mmol) was added. Itwas stirred at room temperature overnight. Water and EtOAc were added.The organic phase was separated, washed with 1 N HCl, then with 5%NaHCO3, dried over Na2SO4, concentrated in vacuo to give2-(1H-benzo[d][1,2,3]triazol-1-yloxy)-4-(1-methyl-1H-indol-4-ylamino)pyrimidine-5-carboxamide(378 mg). MS 401.4 (M+H)

Step 4: To a solution of2-(1H-benzo[d][1,2,3]triazol-1-yloxy)-4-(1-methyl-1H-indol-4-ylamino)pyrimidine-5-carboxamide(62 mg, 0.15 mmol) in NMP (1 ml) was added4-(2-(pyrrolidin-1-yl)ethoxy)aniline (31 mg, 0.15 mmol) andp-toluenesulfonic acid hydrate (28 mg, 0.15 mmol). It was heated at 100°C. for 4 h, and was purified by preparative HPLC to give4-(1-methyl-1H-indol-4-ylamino)-2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenylamino)pyrimidine-5-carboxamide(32 mg). MS found for C26H29N7O2 as (M+H)⁺ 472.4. UV: λ=216.9, 257.0.

Example 1524-(1H-indol-4-ylamino)-2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 151. MS found for C25H27N7O2 as (M+H)⁺ 458.4. UV:λ=214.5, 249.9, 280.7.

Example 1534-(2-methyl-2H-indazol-7-ylamino)-2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 151. MS found for C25H28N8O2 as (M+H)⁺ 473.4. UV:λ=211.0, 275.9.

Example 1544-(1-methyl-1H-indazol-7-ylamino)-2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 151. MS found for C25H28N8O2 as (M+H)+ 473.4. UV:λ=208.6, 286.6.

Example 1552-(4-(1H-imidazol-1-ylphenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed above. MS found for C17H17N7O as (M+H)⁺ 336.3. UV: λ=245.2,321.1.

Example 1562-(4-(2-(pyrrolidin-1-yl)ethoxy)phenylamino)-4-(m-tolylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using meta-anisidine in place of cyclobutylamine.MS found for C₂₄H₂₈N₆O₂ as (M+H)⁺ 433.3. UV: λ=283.

Example 1574-(3,5-dimethylphenylamino)-2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using 3,5-dimethylaniline in place ofcyclobutylamine. MS found for C₂₅H₃₀N₆O₂ as (M+H)⁺ 447.3. UV: λ=283.

Example 1584-(3-methoxyphenylamino)-2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with meta-anisidine in place of cyclobutylamine.MS found for C₂₄H₂₈N₆O₃ as (M+H)⁺ 449.3. UV: λ=282.

Example 1612-(4-(2-hydroxyethylcarbamoyl)phenylamino)-4-(2,2,2-trifluoroethylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed Scheme 1 using trifluoroethylamine in place ofcyclobutylamine. MS found for C₁₆H₁₇F₃N₆O₃ as (M+H)⁺ 399.3.

Example 1624-(4-(2-morpholinoethoxy)phenylamino)-2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 2. MS found for C₂₉H₃₇N₇O₄ as (M+H)⁺ 548.3. UV:λ=283.

Example 1632-(4-(2-(piperidin-1-yl)ethoxy)phenylamino)-4-(m-tolylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using meta-toluidine in place of cyclobutylamine.MS found for C₂₅H₃₀N₆O₂ as (M+H)⁺ 447.3.

Example 1652-(4-(2-methoxyethoxy)phenylamino)-4-(m-tolylamino)pyrimidine-5-carboxamide

MS found for C₂₁H₂₃N₅O₃ as (M+H)⁺ 394.3. UV: λ=285.

Example 1662-(4-(3-hydroxypropylcarbamoyl)phenylamino)-4-(2,2,2-trifluoroethylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using trifluoroethylamine in place ofcyclobutylamine. MS found for C₁₇H₁₉F₃N₆O₃ as (M+H)⁺ 413.4. ¹H NMR(CD₃OD, 400 MHz): δ 8.59 (s, 1H), 7.88 (d, 2H), 7.77 (d, 2H), 4.42 (q,2H), 3.64 (t, 2H), 3.49 (t, 2H), 1.83 (m, 2H).

Example 1672-(4-(5,6-dihydro-4H-1,3-oxazin-2-yl)phenylamino)-4-(2,2,2-trifluoroethylamino)pyrimidine-5-carboxamide

The above compound was prepared by treating Example 166 withdiethylaminosulfurtrifluoride and DIPEA in dichloromethane. MS found forC₁₇H₁₇F₃N₆O₂ as (M+H)⁺ 395.2. UV: λ=221, 310. ¹H NMR (CD₃OD, 400 MHz): δ8.60 (s, 1H), 7.97 (dd, 2H), 7.93 (dd, 2H), 4.33 (q, 2H), 3.73 (t, 2H),253 (m, 2H), 1.21 (t, 2H).

Example 168 4-(cyclobutylamino)-2-(p-tolylamino)pyrimidine-5-carboxamide

The above compound was prepared using para-toluidine and a proceduresimilar to that described in Scheme 1. MS found for C₁₆H₁₉N₅O as (M+H)⁺298.0.

Example 1692-(4-carbamoylphenylamino)-4-(cyclobutylamino)pyrimidine-5-carboxamide

The above compound was prepared using 4-aminobenzamide using a proceduresimilar to that described in Scheme 1. MS found for C₁₆H₁₈N₆O₂ as (M+H)⁺327.0.

Example 1704-(cyclobutylamino)-2-(4-(N-methylcyclopropanecarboxamido)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared usingN-(4-aminophenyl)-N-methylcyclopropanecarboxamide (synthesized fromN-methyl 4-nitroaniline in two steps) using a procedure similar to thatdescribed in Scheme 1. MS found for C₂₀H₂₄N₆O₂ as (M+H)⁺ 381.0.

Example 1712-(3-chloro-4-(N-methylacetamido)phenylamino)-4-(cyclobutylamino)pyrimidine-5-carboxamide

The above compound was prepared usingN-(4-amino-2-chlorophenyl)-N-methylacetamide (prepared from2-chloro-4-nitroaniline in three steps) using a procedure similar tothat described in Scheme 1. MS found for C₁₇H₁₉ClN₆O₂ as (M+H)⁺ 375.0,377.0.

Example 1722-(3-chloro-4-(N-methylacetamido)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using an intermediate synthesized asdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylaminein Step 4, and N-(4-amino-2-chlorophenyl)-N-methylacetamide (preparedfrom 2-chloro-4-nitroaniline in three steps) using a procedure similarto that described in Scheme 1. MS found for C₁₇H₁₉ClN₆O₂ as (M+H)⁺375.0, 377.0. ¹H NMR (CD₃OD, 400 MHz): δ 8.43 (d, 1H), 8.20 (s, 1H),7.61 (dd, 1H), 7.43 (d, 1H), 3.18 (s, 3H), 2.98 (m, 1H), 1.82 (s, 3H),1.01 (m, 2H), 0.73 (m, 2H).

Example 1734-(cyclopropylamino)-2-(4-(N-methylcyclopropanecarboxamido)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using an intermediate synthesized asdescribed in Example 139 with cyclopropylamine in place ofcyclobutylamine in Step 4, andN-(4-aminophenyl)-N-methylcyclopropanecarboxamide (synthesized fromN-methyl 4-nitroaniline in two steps) using a procedure similar to thatdescribed in Scheme 1. MS found for C₁₉H₂₂N₆O₂ as (M+H)⁺ 367.1.

Example 1744-(cyclopropylamino)-2-(4-(4-(2-hydroxyethyl)piperazin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine.MS found for C₂₀H₂₇N₇O₂ as (M+H)⁺ 398.3.

Example 1754-(cyclopropylamino)-2-(4-(4-(methylcarbamoyl)piperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place of cyclobutylamineand an aniline derived from 4-fluoronitrobenzene and ethylisonipecotate. MS found for C₂₁H₂₇N₇O₂ as (M+H)⁺ 410.0.

Example 1764-(cyclopropylamino)-2-(4-(4-(dimethylcarbamoyl)piperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place of cyclobutylamineand an aniline derived from 4-fluoronitrobenzene and ethylisonipecotate. MS found for C₂₂H₂₉N₇O₂ as (M+H)⁺ 424.0.

Example 1774-(cyclopropylamino)-2-(4-(4-(dimethylcarbamoyl)piperazin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared by treating4-(4-nitrophenyl)piperazine-1-carboxamide with sodium hydride and methyliodide, then reducing the nitro using hydrogenation. The aniline wasthen coupled using a procedure similar to that described in Scheme 1with cyclopropylamine in place of cyclobutylamine. MS found forC₂₁H₂₈N₈O₂ as (M+H)⁺ 425.2.

Example 1782-(3-chloro-4-morpholinophenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine.MS found for C₁₈H₂₁N₆O₂Cl as (M+H)⁺ 389.0, 391.0.

Example 1794-(cyclopropylamino)-2-(4-(3,3-difluoropyrrolidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamineand an aniline derived from 3,3-difluoropyrrolidine and4-fluoronitrobenzene in two steps. MS found for C₁₈H₂₀N₆OF₂ as (M+H)⁺375.0.

Example 1804-(cyclopropylamino)-2-(4-(methylcarbamoyl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamineand an aniline derived from 4-nitrobenzoylchloride and methylamine. MSfound for C₁₆H₁₈N₆O₂ as (M+H)⁺ 327.0.

Example 1814-(cyclopropylamino)-2-(4-(dimethylcarbamoyl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamineand an aniline derived from 4-nitrobenzoylchloride and dimethylamine. MSfound for C₁₇H₂₀N₆O₂ as (M+H)⁺ 341.0.

Example 1824-(cyclopropylamino)-2-(4-(4-(methylsulfonyl)piperazine-1-carbonyl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamineand an aniline derived from 4-nitrobenzoylchloride. MS found forC₂₀H₂₅N₇O₄S as (M+H)⁺ 460.1.

Example 1834-(cyclopropylamino)-2-(4-(thiazol-4-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine.MS found for C₁₇H₁₆N₆OS as (M+H)⁺ 353.2.

Example 1842-(4-(1H-pyrazol-1-yl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine.MS found for C₁₇H₁₇N₇O as (M+H)⁺ 336.3.

Example 1852-(4-(1H-tetrazol-1-yl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine.MS found for C₁₅H₁₅N₉O as (M+H)⁺ 338.2.

Example 1864-(cyclopropylamino)-2-(4-(thiophen-2-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine.MS found for C₁₈H₁₇N₅OS as (M+H)⁺ 352.2.

Example 1874-(cyclopropylamino)-2-(4-formamido-3-hydroxyphenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamineusing 6-aminobenzoxazole which subsequently hydrolyzed duringpurification affording the titled compound. MS found for C₁₅H₁₆N₆O₃ as(M+H)⁺ 329.2.

Example 1884-(cyclopropylamino)-2-(4-(pyridin-2-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine.MS found for C₁₉H₁₈N₆O as (M+H)⁺ 347.3.

Example 1892-(4-(1-acetyl-1,2,3,6-tetrahydropyridin-4-yl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine.MS found for C₂₁H₂₄N₆O₂ as (M+H)⁺ 393.3.

Example 190(R)-4-(cyclopropylamino)-2-(4-(2-(methoxymethyl)pyrrolidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamineand using an aniline prepared in two steps from 4-fluoronitrobenzene and(R)-2-methoxymethylpyrrolidine. MS found for C₂₀H₂₆N₆O₂ as (M+H)⁺ 383.3.

Example 191(S)-4-(cyclopropylamino)-2-(4-(2-(methoxymethyl)pyrrolidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 190 using (S)-2-methoxymethylpyrrolidine in placeof the (R)-isomer. MS found for C₂₀H₂₆N₆O₂ as (M+H)⁺ 383.3. ¹H NMR(CD₃OD, 400 MHz): δ 8.21 (s, 1H), 7.38 (broad s, 2H), 6.78 (d, 2H), 3.89(m, 1H), 3.49 (m, 2H), 3.33 (s, 3H), 3.18 (m, 2H), 3.03 (m, 1H), 2.03(m, 4H), 0.93 (m, 2H), 0.72 (m, 2H).

Example 1924-(cyclopropylamino)-2-(4-(pyridin-3-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine.MS found for C₁₉H₁₈N₆O as (M+H)⁺ 347.3.

Example 1934-(cyclopropylamino)-2-(4-(ethylsulfonyl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine.MS found for C₁₆H₁₉N₅O₃S as (M+H)⁺ 362.1.

Example 1942-(1H-indol-6-ylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine.MS found for C₁₆H₁₆N₆O as (M+H)⁺ 309.2.

Example 1951-(4-(5-carbamoyl-4-(cyclopropylamino)pyrimidin-2-ylamino)phenyl)piperidine-4-carboxylicacid

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamineand an aniline derived from ethyl isonipecotate and4-fluoronitrobenzene. MS found for C₂₀H₂₄N₆O₃ as (M+H)⁺ 397.2.

Example 1964-(cyclopropylamino)-2-(4-(4-(methylsulfonyl)-1,4-diazepan-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamineand an aniline derived from Boc protected homopiperazine and4-fluoronitrobenzene. MS found for C₂₀H₂₇N₇O₃S as (M+H)⁺ 446.2.

Example 1974-(cyclopropylamino)-2-(4-(3,5-dimethyl-1H-pyrazol-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine.MS found for C₁₉H₂₁N₇O as (M+H)⁺ 364.2.

Example 198(S)-2-(4-(2-carbamoylpyrrolidin-1-yl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamineand an aniline derived from (S)-prolinamide and 4-fluoronitrobenzene. MSfound for C₁₉H₂₃N₇O₂ as (M+H)⁺ 382.0.

Example 1994-(cyclopropylamino)-2-(4-(cyclopropylcarbamoyl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine,using an aniline derived from 4-nitrobenzoyl chloride in two steps. MSfound for C₁₈H₂₀N₆O₂ as (M+H)⁺ 353.0.

Example 2002-(4-(cyclobutylcarbamoyl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 199. MS found for C₁₉H₂₂N₆O₂ as (M+H)⁺ 367.3.

Example 201(S)-4-(cyclopropylamino)-2-(4-(2-(methylcarbamoyl)pyrrolidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1, with cyclopropylamine in place of cyclobutylamineand an aniline derived from (S)-proline and 4-fluoronitrobenzene. MSfound for C₂₀H₂₅N₇O₂ as (M+H)⁺ 396.0.

Example 202(S)-4-(cyclopropylamino)-2-(4-(2-(dimethylcarbamoyl)pyrrolidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201. MS found for C₂₁H₂₇N₇O₂ as (M+H)⁺ 410.0.

Example 2034-(cyclopropylamino)-2-(4-(2-hydroxyethylcarbamoyl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place of cyclobutylamineand an aniline derived from 4-nitrobenzoyl chloride and ethanolamine. MSfound for C₁₇H₂₀N₆O₃ as (M+H)⁺ 357.0.

Example 2044-(cyclopropylamino)-2-(4-(3-hydroxypropylcarbamoyl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place of cyclobutylamineand an aniline derived from 4-nitrobenzoyl chloride and propanolamine.MS found for C₁₈H₂₂N₆O₃ as (M+H)⁺ 371.0.

Example 205(R)-4-(cyclopropylamino)-2-(4-(3-(methylcarbamoyl)piperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201. MS found for C₂₁H₂₇N₇O₂ as (M+H)⁺ 410.0.

Example 206(R)-4-(cyclopropylamino)-2-(4-(3-(dimethylcarbamoyl)piperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201. MS found for C₂₂H₂₉N₇O₂ as (M+H)⁺ 424.0.

Example 207(S)-4-(cyclopropylamino)-2-(4-(3-(methylcarbamoyl)piperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201. MS found for C₂₁H₂₇N₇O₂ as (M+H)⁺ 410.2.

Example 208(S)-4-(cyclopropylamino)-2-(4-(3-(dimethylcarbamoyl)piperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201. MS found for C₂₂H₂₉N₇O₂ as (M+H)⁺ 424.2.

Example 209(S)-4-(cyclopropylamino)-2-(4-(2-(methylcarbamoyl)piperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201. MS found for C₂₁H₂₇N₇O₂ as (M+H)⁺ 410.2.

Example 210(S)-4-(cyclopropylamino)-2-(4-(2-(dimethylcarbamoyl)piperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201. MS found for C₂₂H₂₉N₇O₂ as (M+H)⁺ 424.2.

Example 2114-(cyclopropylamino)-2-(quinoxalin-6-ylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₁₆H₁₅N₇O as (M+H)⁺ 322.1.

Example 212(S)-2-(4-(2-carbamoylazetidin-1-yl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201. MS found for C₁₈H₂₁N₇O₂ as (M+H)⁺ 368.1.

Example 2134-(cyclopropylamino)-2-(4-(4-hydroxypiperidin-1-ylsulfonyl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1, using cyclopropylamine in place ofcyclobutylamine and an aniline prepared from 4-nitrobenzenesulfonylchloride and 4-hydroxypoperidine. MS found for C₁₉H₂₄N₆O₄S as (M+H)⁺433.0.

Example 2142-(4-(cyclopropyl(methyl)carbamoyl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₁₉H₂₂N₆O₂ as (M+H)⁺ 367.0.

Example 2152-(4-(cyclobutyl(methyl)carbamoyl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₂₀H₂₄N₆O₂ as (M+H)⁺ 381.0.

Example 2162-(4-(2-aminopyridin-4-yl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₁₉H₁₉N₇O as (M+H)⁺ 362.4.

Example 217(R)-4-(cyclopropylamino)-2-(4-(2-(methylcarbamoyl)pyrrolidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201. MS found for C₂₀H₂₅N₇O₂ as (M+H)⁺ 396.0.

Example 218(R)-4-(cyclopropylamino)-2-(4-(2-(dimethylcarbamoyl)pyrrolidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201. MS found for C₂₁H₂₇N₇O₂ as (M+H)⁺ 410.0.

Example 2194-(cyclopropylamino)-2-(4-(3-(methylcarbamoyl)pyrrolidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201. MS found for C₂₀H₂₅N₇O₂ as (M+H)⁺ 396.5.

Example 2202-(4-((2S,4S)-2-carbamoyl-4-hydroxypyrrolidin-1-yl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201. MS found for C₁₉H₂₃N₇O₃ as (M+H)⁺ 398.5.

Example 2214-(cyclopropylamino)-2-(4-((2S,4S)-4-hydroxy-2-(methylcarbamoyl)pyrrolidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201. MS found for C₂₀H₂₅N₇O₃ as (M+H)⁺ 412.5.

Example 2224-(cyclobutylamino)-2-(4-(dimethylamino)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using N,N-dimethylbenzene-1,4-diamineusing a procedure similar to that described in Scheme 1. MS found forC₁₇H₂₂N₆O as (M+H)⁺ 327.0.

Example 2232-(4-acetamidophenylamino)-4-(cyclobutylamino)pyrimidine-5-carboxamide

The above compound was prepared using N-(4-aminophenyl)acetamide using aprocedure similar to that described in Scheme 1. MS found for C₁₇H₂₀N₆O₂as (M+H)⁺ 341.0.

Example 2242-(1H-indazol-5-ylamino)-4-(cyclobutylamino)pyrimidine-5-carboxamide 16

The above compound was prepared using 1H-indazol-5-amine using aprocedure similar to that described in Scheme 1. MS found for C₁₆H₁₇N₇Oas (M+H)⁺ 324.0.

Example 2252-(1H-indazol-6-ylamino)-4-(cyclobutylamino)pyrimidine-5-carboxamide

The above compound was prepared using 1H-indazol-6-amine using aprocedure similar to that described in Scheme 1. MS found for C₁₆H₁₇N₇Oas (M+H)⁺ 324.0. ¹H NMR (CD₃OD, 400 MHz): δ 8.44 (s, 1H), 8.13 (s, 1H),7.99 (s, 1H), 7.88 (d, 1H), 7.27 (m, 2H), 4.63 (m, 1H), 2.48 (m, 2H),2.13 (m, 2H), 1.92 (m, 2H)

Example 2264-(cyclobutylamino)-2-(4-methoxyphenylamino)pyrimidine-5-carboxamide

The above compound was prepared using p-anisidine using a proceduresimilar to that described in Scheme 1. MS found for C₁₆H₁₉N₅O₂ as (M+H)⁺314.0.

Example 2274-(cyclobutylamino)-2-(4-(diethylamino)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using N,N-diethyl phenylenediamine usinga procedure similar to that described in Scheme 1. MS found forC₁₉H₂₆N₆O as (M+H)⁺ 355.2.

Example 228 4-(cyclobutylamino)-2-(phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using aniline using a procedure similarto that described in Scheme 1. MS found for C₁₅H₁₇N₅O as (M+H)⁺ 284.0.

Example 2294-(cyclobutylamino)-2-(4-(N-methylpropionamido)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared usingN-(4-aminophenyl)-N-methylpropionamide (synthesized from N-methyl4-nitroaniline in two steps) using a procedure similar to that describedin Scheme 1. MS found for C₁₉H₂₄N₆O₂ as (M+H)⁺ 369.0.

Example 2304-(cyclopropylamino)-2-(4-(N-methylpropionamido)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using an intermediate synthesized asdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamineand N-(4-aminophenyl)-N-methylpropionamide (synthesized from N-methyl4-nitroaniline in two steps). MS found for C₁₈H₂₂N₆O₂ as (M+H)⁺ 355.0.

Example 2314-(cyclopropylamino)-2-(4-(N-methylacetamido)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using an intermediate synthesized asdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamineand N-(4-aminophenyl)-N-methylacetamide (which was prepared fromN-methyl 4-nitroaniline in two steps) MS found for C₁₇H₂₀N₆O₂ as (M+H)⁺341.0.

Example 232 isopropyl4-(5-carbamoyl-4-(cyclopropylamino)pyrimidin-2-ylamino)phenyl(methyl)carbamate

The above compound was prepared using an intermediate synthesized asdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamineand isopropyl 4-aminophenyl(methyl)carbamate (synthesized from N-methyl4-nitroaniline in two steps). MS found for C₁₉H₂₄N₆O₃ as (M+H)⁺ 385.0.

Example 2334-(cyclopropylamino)-2-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine.MS found for C₁₆H₁₇N₅O₃ as (M+H)⁺ 328.2.

Example 2344-(cyclopropylamino)-2-(4-morpholinophenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine.MS found for C₁₈H₂₂N₆O₂ as (M+H)⁺ 355.0. ¹H NMR (DMSO-d₆, 400 MHz): δ8.24 (s, 1H), 7.50 (broad s, 2H), 7.06 (d, 2H), 3.84 (m, 4H), 3.21 (m,4H), 0.92 (m, 2H), 0.71 (m, 2H).

Example 2354-(cyclopropylamino)-2-(4-(piperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamine.MS found for C₁₉H₂₄N_(x6)O as (M+H)⁺ 353.2.

Example 2364-(cyclopropylamino)-2-(4-(4-hydroxypiperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamineand an aniline derived from 4-fluoronitrobenzene and4-hydroxypiperidine. MS found for C₁₉H₂₄N₆O₂ as (M+H)⁺ 369.0.

Example 2374-(cyclopropylamino)-2-(4-(4-fluoropiperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamineand an aniline derived from 4-fluoronitrobenzene and 4-fluoropiperidine.MS found for C₁₉H₂₃N₆OF as (M+H)⁺ 371.0.

Example 2384-(cyclopropylamino)-2-(4-(4-methylpiperazin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamineand an aniline derived from 4-fluoronitrobenzene and 1-methylpiperazine.MS found for C₁₉H₂₅N₇O as (M+H)⁺ 368.3.

Example 2394-(cyclopropylamino)-2-(4-(3,3-difluoropiperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamineand an aniline derived from 4-fluoronitrobenzene and3,3-difluoropiperidine. MS found for C₁₉H₂₂N₆OF₂ as (M+H)⁺ 389.0.

Example 2402-(4-(4-carbamoylpiperidin-1-yl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamineand an aniline prepared from 4-fluoronitrobenzene and isonipecotamidefollowed by reduction using hydrogen and Pd/C in methanol. MS found forC₂₀H₂₅N₇O₂ as (M+H)⁺ 396.2.

Example 2412-(4-(4-carbamoylpiperazin-1-yl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using an aniline prepared from4-piperazinylnitrobenzene in two steps. MS found for C₁₉H₂₄N₈O₂ as(M+H)⁺ 397.2.

Example 2424-(cyclopropylamino)-2-(4-(4,4-difluoropiperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 with cyclopropylamine in place of cyclobutylamineand an aniline derived from 4-fluoronitrobenzene and4,4-difluoropiperidine. MS found for C₁₉H₂₂N₆OF₂ as (M+H)⁺ 389.0.

Example 2444-(cyclopropylamino)-2-(4-(methylsulfonyl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₁₅H₁₇N₅O₃S as (M+H)⁺ 348.1.

Example 2454-(cyclopropylamino)-2-(4-(methylthio)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₁₅H₁₇N₅OS as (M+H)⁺ 316.1.

Example 2464-(cyclopropylamino)-2-(4-((4-(methylsulfonyl)piperazin-1-yl)methyl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₂₀H₂₇N₇O₃S as (M+H)⁺ 446.3.

Example 2474-(cyclopropylamino)-2-(4-(piperazin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₁₈H₂₃N₇O as (M+H)⁺ 354.2.

Example 2482-(4-(1H-1,2,4-triazol-1-yl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₁₆H₁₆N₈O as (M+H)⁺ 352.2.

Example 2492-(1H-indol-5-ylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₁₆H₁₆N₆O as (M+H)⁺ 309.2.

Example 2502-(1H-indazol-5-ylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₁₅H₁₅N₇O as (M+H)⁺ 310.2.

Example 2514-(cyclopropylamino)-2-(4-(oxazol-5-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₁₇H₁₆N₆O₂ as (M+H)⁺ 337.1.

Example 2522-(1H-indazol-6-ylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₁₅H₁₅N₇O as (M+H)⁺ 310.2.

Example 2534-(cyclopropylamino)-2-(quinolin-6-ylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₁₇H₁₆N₆O as (M+H)⁺ 321.2.

Example 254(R)-2-(4-(3-carbamoylpiperidin-1-yl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201. MS found for C₂₀H₂₅N₇O₂ as (M+H)⁺ 396.0.

Example 255(S)-2-(4-(3-carbamoylpiperidin-1-yl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201. MS found for C₂₀H₂₅N₇O₂ as (M+H)⁺ 396.2.

Example 2564-(cyclobutylamino)-2-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₁₇H₁₉N₅O₃ as (M+H)⁺ 342.2.

Example 2574-(cyclobutylamino)-2-(4-(piperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place of cyclobutylamineand an aniline derived from piperidine and 4-fluoronitrobenzene. MSfound for C₂₀H₂₆N₆O as (M+H)⁺ 367.3.

Example 2584-(cyclobutylamino)-2-(4-(4-hydroxypiperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place of cyclobutylamineand an aniline derived from 4-hydroxypiperidine and4-fluoronitrobenzene. MS found for C₂₀H₂₆N₆O₂ as (M+H)⁺ 383.3.

Example 2594-(cyclobutylamino)-2-(4-(4-fluoropiperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place of cyclobutylamineand an aniline derived from 4-fluoropiperidine and 4-fluoronitrobenzene.MS found for C₂₀H₂₅N₆OF as (M+H)⁺ 385.0.

Example 2604-(cyclobutylamino)-2-(4-morpholinophenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place of cyclobutylamineand an aniline derived from 4-morpholine and 4-fluoronitrobenzene. MSfound for C₁₉H₂₄N₆O₂ as (M+H)⁺ 369.0.

Example 2614-(cyclobutylamino)-2-(4-(4-methylpiperazin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place of cyclobutylamineand an aniline derived from 1-methylpiperazine and 4-fluoronitrobenzene.MS found for C₂₀H₂₇N₇O as (M+H)⁺ 382.3.

Example 2624-(cyclobutylamino)-2-(4-(3,3-difluoropiperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place of cyclobutylamineand an aniline derived from 3,3-difluoropiperidine and4-fluoronitrobenzene. MS found for C₂₀H₂₄N₆OF₂ as (M+H)⁺ 403.0.

Example 2634-(cyclobutylamino)-2-(4-(4,4-difluoropiperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place of cyclobutylamineand an aniline derived from 4,4-difluoropiperidine and4-fluoronitrobenzene. MS found for C₂₀H₂₄N₆OF₂ as (M+H)⁺ 403.0.

Example 264(R)-2-(4-(2-carbamoylpyrrolidin-1-yl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201. MS found for C₁₉H₂₃N₇O₂ as (M+H)⁺ 382.0.

Example 2652-(4-((2S,4R)-2-carbamoyl-4-hydroxypyrrolidin-1-yl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201.

Example 2664-(cyclopropylamino)-2-(4-((2S,4R)-4-hydroxy-2-(methylcarbamoyl)pyrrolidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201. MS found for C₂₀H₂₅N₇O₃ as (M+H)⁺ 412.5.

Example 2674-(cyclopropylamino)-2-(4-((2S,4R)-2-(dimethylcarbamoyl)-4-hydroxypyrrolidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201. MS found for C₂₁H₂₇N₇O₃ as (M+H)⁺ 426.5.

Example 2684-(cyclobutylamino)-2-(4-(4-(methylcarbamoyl)piperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using an aniline derived from ethyl isonipecotateand 4-fluoronitrobenzene. MS found for C₂₂H₂₉N₇O₂ as (M+H)⁺ 424.0.

Example 2694-(cyclobutylamino)-2-(4-(4-(dimethylcarbamoyl)piperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 in Scheme 1 using an aniline derived from ethylisonipecotate and 4-fluoronitrobenzene. MS found for C₂₃H₃₁N₇O₂ as(M+H)⁺ 438.3.

Example 2704-(cyclobutylamino)-2-(4-(4-(2-hydroxyethyl)piperazin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using an aniline derived from1-hydroxyethylpiperazine and 4-fluoronitrobenzene. MS found forC₂₁H₂₉N₇O₂ as (M+H)⁺ 412.3.

Example 2712-(3-chloro-4-morpholinophenylamino)-4-(cyclobutylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1. MS found for C₁₉H₂₃N_(x6)O₂Cl as (M+H)⁺ 403.0,405.0.

Example 2724-(cyclobutylamino)-2-(4-(3,3-difluoropyrrolidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using an aniline derived from3,3-difluoropyrrolidine and 4-fluoronitrobenzene. MS found forC₁₉H₂₂N₆OF₂ as (M+H)⁺ 389.0.

Example 2734-(cyclobutylamino)-2-(4-(methylcarbamoyl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1. MS found for C₁₇H₂₀N₆O₂ as (M+H)⁺ 341.0.

Example 2744-(cyclobutylamino)-2-(4-(dimethylcarbamoyl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1. MS found for C₁₈H₂₂N₆O₂ as (M+H)⁺ 355.2.

Example 275(S)-4-(methylamino)-2-(4-(2-(methylcarbamoyl)piperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201 using methylamine in place of cyclobutylaminein the synthesis of the starting material described in Scheme 1. MSfound for C₁₉H₂₅N₇O₂ as (M+H)⁺ 384.3. UV: λ=208, 276.

Example 276(S)-4-(ethylamino)-2-(4-(2-(methylcarbamoyl)piperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201 using ethylamine in place of cyclobutylamine inthe synthesis of the starting material described in Scheme 1. MS foundfor C₂₀H₂₇N₇O₂ as (M+H)⁺ 398.3. UV: λ=203, 272.

Example 277(S)-2-(4-(2-(methylcarbamoyl)piperidin-1-yl)phenylamino)-4-(prop-2-ynylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201 using propargylamine in place ofcyclobutylamine in the synthesis of the starting material described inScheme 1 with propargylamine in place of methylamine. MS found forC₂₁H₂₅N₇O₂ as (M+H)⁺ 408.3. UV: λ=207, 273.

Example 278(S)-4-(cyclopropylmethylamino)-2-(4-(2-(methylcarbamoyl)piperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201 using cyclopropylmethylamine in place ofcyclobutylamine in the synthesis of the starting material described inScheme 1. MS found for C₂₂H₂₉N₇O₂ as (M+H)⁺ 424.4. UV: λ=204, 260.

Example 279(R)-2-(4-(3-carbamoylpiperidin-1-yl)phenylamino)-4-(2,2,2-trifluoroethylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201 using trifluoroethylamine in place ofcyclobutylamine in the synthesis of the starting material described inScheme 1. MS found for C₁₉H₂₂F₃N₇O₂ as (M+H)⁺ 438.3.

Example 280(R)-2-(4-(3-(methylcarbamoyl)piperidin-1-yl)phenylamino)-4-(2,2,2-trifluoroethylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201 using trifluoroethylamine in place ofcyclobutylamine in the synthesis of the starting material described inScheme 1. MS found for C₂₀H₂₄F₃N₇O₂ as (M+H)⁺ 452.3.

Example 281(S)-2-(4-(2-carbamoylpiperidin-1-yl)phenylamino)-4-(2,2,2-trifluoroethylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201 using trifluoroethylamine in place ofcyclobutylamine in the synthesis of the starting material described inScheme 1. MS found for C₁₉H₂₂F₃N₇O₂ as (M+H)⁺ 438.3.

Example 282(S)-2-(4-(2-(dimethylcarbamoyl)piperidin-1-yl)phenylamino)-4-(2,2,2-trifluoroethylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201 using trifluoroethylamine in place ofcyclobutylamine in the synthesis of the starting material described inScheme 1. MS found for C₂₁H₂₆F₃N₇O₂ as (M+H)⁺ 466.4.

Example 2832-(4-methoxyphenylamino)-4-(2,2,2-trifluoroethylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1, using trifluoroethylamine in place ofcyclobutylamine and by using para-anisidine in the last step. MS foundfor C₁₄H₁₄F₃N₅O₂ as (M+H)⁺ 342.2. UV: λ=217.

Example 2842-(4-carbamoylphenylamino)-4-(2,2,2-trifluoroethylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using trifluoroethylamine in place ofcyclobutylamine and by using 4-aminobenzamide in the last step. MS foundfor C₁₄H₁₃F₃N₆O₂ as (M+H)⁺ 355.2. UV: λ=200, 290.

Example 285(S)-4-(tert-butylamino)-2-(4-(2-(methylcarbamoyl)piperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201 using tert-butylamine in place ofcyclobutylamine in the synthesis of the starting material described inScheme 1. MS found for C₂₂H₃₁N₇O₂ as (M+H)⁺ 426.3.

Example 286(S)-4-(2-methoxyethylamino)-2-(4-(2-(methylcarbamoyl)piperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201 using 2-methoxyethylamine in place ofcyclobutylamine in the synthesis of the starting material described inScheme 1. MS found for C₂₁H₂₉N₇O₃ as (M+H)⁺ 428.3. UV: λ=202, 268.

Example 287(S)-4-(cyclopentylamino)-2-(4-(2-(methylcarbamoyl)piperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201 using cyclopentylamine in place ofcyclobutylamine in the synthesis of the starting material described inScheme 1. MS found for C₂₃H₃₁N₇O₂ as (M+H)⁺ 438.3. UV: λ=203, 262.

Example 2884-(cyclopropylamino)-2-(4-ethoxyphenylamino)pyrimidine-5-carboxamide

Intermediate 361.1 stirred with 4-ethoxyaniline 361.2 [CAS156-43-4] andpTSA in NMP. Reaction heated at 120° C. for 2 h in a sealed tube. Thereaction cooled, turned slightly acidic with aqueous TFA, and purifiedby reverse phase preparative HPLC to afford the title compound. MS foundfor C₁₆H₁₉N₅O₂ as (M+H)⁺ 314.3. λ=275 nm.

Example 2894-(cyclopropylamino)-2-(4-phenoxyphenylamino)pyrimidine-5-carboxamide

Intermediate 361.1 stirred with 4-phenoxyaniline [CAS 139-59-3] and pTSAin NMP as described in example 288. Reverse phase preparative HPLCafforded the title compound. MS found for C₂₀H₁₉N₅O₂ as (M+H)⁺ 362.2. UVλ=277 nm.

Example 2904-(cyclopropylamino)-2-(4-(trifluoromethoxy)phenylamino)pyrimidine-5-carboxamide

Intermediate 361.1 stirred with 4-(trifluoromethoxy)aniline [CAS461-82-5] and pTSA in NMP as described in example 288. Reverse phasepreparative HPLC afforded the title compound. MS found for C₁₅H₁₄F₃N₅O₂as (M+H)⁺ 354.2. UV λ=265 nm.

Example 2914-(cyclopropylamino)-2-(4-(pyridin-3-yloxy)phenylamino)pyrimidine-5-carboxamide

Intermediate 361.1 stirred with 4-(3-Pyridyloxy)aniline [CAS 80650-45-9]and pTSA in NMP as described in example 288. Reverse phase preparativeHPLC afforded the title compound. MS found for C₁₉H₁₈N₆O₂ as (M+H)⁺363.3. UV λ=205, 267 nm.

Example 2922-(4-(2-cyanoethylsulfonyl)phenylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

Intermediate 361.1 stirred with 3-(4-aminophenyl)sulfonyl propanenitrile[CAS 84362-27-6] and pTSA in NMP as described in example 288. Reversephase preparative HPLC afforded the title compound. MS found forC₁₇H₁₈N₆O₃S as (M+H)⁺ 387.2. UV λ=290 nm.

Example 2934-(cyclopropylamino)-2-(4-isobutoxyphenylamino)pyrimidine-5-carboxamide

Intermediate 361.1 stirred with 4-(2-methylpropoxy)aniline hydrochloride[CAS 1050161-26-6] and pTSA in NMP as described in example 288. Reversephase preparative HPLC afforded the title compound. MS found forC₁₈H₂₃N₅O₂ as (M+H)⁺ 342.4. UV λ=280 nm.

Example 2944-(cyclopropylamino)-2-(4-(thiazol-4-ylmethylsulfonyl)phenylamino)pyrimidine-5-carboxamide

Intermediate 361.1 stirred with 4-(thiazol-4-ylmethylsulfonyl)anilineand pTSA in NMP as described in example 288. Reverse phase preparativeHPLC afforded the title compound. MS found for C₁₈H₁₈N₆O₃S₂ as (M+H)⁺431.2. UV λ=291 nm.

Example 295(S)-4-(benzylamino)-2-(4-(2-(methylcarbamoyl)piperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201 using benzylamine in place of cyclobutylaminein the synthesis of the starting material described in Scheme 1. MSfound for C₂₅H₂₉N₇O₂ as (M+H)⁺ 460.3. UV: λ=208, 261.

Example 296(S)-4-(isopropylamino)-2-(4-(2-(methylcarbamoyl)piperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201 using isopropylamine in place ofcyclobutylamine in the synthesis of the starting material described inScheme 1. MS found for C₂₁H₂₉N₇O₂ as (M+H)⁺ 412.4. UV: λ=202, 271.

Example 297(S)-2-(4-(2-(methylcarbamoyl)piperidin-1-yl)phenylamino)-4-(2,3,6-trifluorobenzylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201 using 2,3,6-trifluorobenzylamine in place ofcyclobutylamine in the synthesis of the starting material described inScheme 1. MS found for C₂₅H₂₆F₃N₇O₂ as (M+H)⁺ 514.4.

Example 298(S)-4-(2-methoxyethylamino)-2-(4-(2-(methylcarbamoyl)piperidin-1-yl)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 201 using 2-methoxyethylamine in place ofcyclobutylamine in the synthesis of the starting material described inScheme 1. MS found for C₂₂H₃₁N₇O₃ as (M+H)⁺ 442.4. UV: λ=203, 273.

Example 2994-(1-methyl-1H-indazol-4-ylamino)-2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 151. MS found for C25H28N8O2 as (M+H)⁺ 473.4. UV:λ=212.2, 285.4.

Example 3004-(1,2-dimethyl-1H-indol-4-ylamino)-2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 48. MS found for C27H31N7O2 as (M+H)⁺ 486.5. UV:λ=218.6, 258.9.

Example 3014-(4-chloronaphthalen-1-ylamino)-2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 48. MS found for C27H27ClN6O2 as (M+H)⁺ 503.4,505.4 (Cl pattern). UV: λ=222.8, 284.2.

Example 3024-(3-methylcinnolin-5-ylamino)-2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 48. MS found for C26H28N8O2 as (M+H)⁺ 485.4. UV:λ=276.1.

Example 3034-(benzo[d]thiazol-7-ylamino)-2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 48. MS found for C24H25N7O2S as (M+H)⁺ 476.4. UV:λ=216.9, 281.9.

Example 3044-(naphthalen-1-ylamino)-2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 48. MS found for C27H28N6O2 as (M+H)⁺ 469.4. UV:λ=219.2, 280.7.

Example 3054-(4-(methylsulfonyl)phenylamino)-2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 48. MS found for C₂₄H₂₈N₆O₄S as (M+H)⁺ 497.4. UV:λ=292.

Example 3062-(4-(2-(pyrrolidin-1-yl)ethoxy)phenylamino)-4-(2,2,2-trifluoroethylamino)pyrimidine-5-carboxamide

This compound was synthesized utilizing the chemistry described inScheme 1 with trifluoroethylamine in place of cyclobutylamine. MS foundfor C₁₉H₂₃F₃N₆O₂ as (M+H)⁺ 425.3. UV λ=215, 245, 275 nm.

Example 3074-(benzylamino)-2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenylamino)pyrimidine-5-carboxamide

This compound was synthesized utilizing the chemistry described inScheme 1 with benzylamine in place of cyclobutylamine. MS found forC₂₄H₂₈N₆O₂ as (M+H)⁺ 433.4. UV λ=251 nm.

Example 3094-(cyclobutylamino)-2-(2-oxoindolin-5-ylamino)pyrimidine-5-carboxamide

The above compound was prepared using 5-aminoindolin-2-one and a theprocedure described in Scheme 1. MS found for C₁₇H₁₈N₆O₂ as (M+H)⁺339.0.

Example 3104-(cyclobutylamino)-2-(4-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-ylamino)pyrimidine-5-carboxamide

The above compound was prepared usingN7-amino-4-methyl-2H-benzo[b][1,4]oxazin-3(4H)-one (synthesized from2-amino-5-nitrophenol and ethyl bromoacetate in three steps) using aprocedure similar to that described in Scheme 1. MS found for C₁₈H₂₀N₆O₃as (M+H)⁺ 369.0.

Example 3114-(cyclobutylamino)-2-(2-oxo-1,2,3,4-tetrahydroquinolin-6-ylamino)pyrimidine-5-carboxamide

The above compound was prepared using6-amino-3,4-dihydroquinolin-2(1H)-one (prepared by nitration of3,4-dihydroquinolin-2(1H)-one followed by reduction with iron powder)using a procedure similar to that described in Scheme 1. MS found forC₁₈H₂₀N₆O₂ as (M+H)⁺ 353.0

Example 3124-(cyclobutylamino)-2-(2,2,4-trimethyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-ylamino)pyrimidine-5-carboxamide

The above compound was prepared usingN7-amino-4-methyl-2H-benzo[b][1,4]oxazin-3(4H)-one (synthesized from2-amino-5-nitrophenol and ethyl 2-bromo-2-methylpropanoate in threesteps) using a procedure similar to that described in Scheme 1. MS foundfor C₂₀H₂₄N₆O₃ as (M+H)⁺ 397.0.

Example 3134-(cyclobutylamino)-2-(1-methyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-ylamino)pyrimidine-5-carboxamide

The above compound was prepared using6-amino-1-methyl-3,4-dihydroquinolin-2(1H)-one (prepared by nitration of3,4-dihydroquinolin-2(1H)-one followed by methylation and subsequentreduction with iron powder) using a procedure similar to that describedin Scheme 1. MS found for C₁₉H₂₂N₆O₂ as (M+H)⁺ 367.0.

Example 3144-(cyclobutylamino)-2-(4-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-ylamino)pyrimidine-5-carboxamide

The above compound was prepared using6-amino-4-methyl-2H-benzo[b][1,4]oxazin-3(4H)-one (synthesized from2-amino-4-nitrophenol and ethyl bromoacetate in three steps) using aprocedure similar to that described in Scheme 1. MS found for C₁₈H₂₀N₆O₃as (M+H)⁺ 369.0.

Example 3154-(cyclobutylamino)-2-(3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-ylamino)pyrimidine-5-carboxamide

The above compound was prepared using6-amino-2H-benzo[b][1,4]oxazin-3(4H)-one (synthesized from2-amino-4-nitrophenol and ethyl bromoacetate in two steps) using aprocedure similar to that described in Scheme 1. MS found for C₁₇H₁₈N₆O₃as (M+H)⁺ 355.0.

Example 3162-(1H-benzo[d][1,2,3]triazol-6-ylamino)-4-(cyclobutylamino)pyrimidine-5-carboxamide

The above compound was prepared using 1H-benzo[d][1,2,3]triazol-6-amineusing a procedure similar to that described in Scheme 1. MS found forC₁₅H₁₆N₈O as (M+H)⁺ 325.0.

Example 3174-(cyclobutylamino)-2-(2-oxo-2,3-dihydrobenzofuran-5-ylamino)pyrimidine-5-carboxamide

The above compound was prepared using 5-aminobenzofuran-2(3H)-one usinga procedure similar to that described in Scheme 1. MS found forC₁₇H₁₇N₅O₃ as (M+H)⁺ 340.0. ¹H NMR (DMSO-d₆, 400 MHz): δ 8.41 (s, 1H),8.05 (s, 1H) 7.88 (d, 1H) 7.78 (d, 1H), 4.59 (m, 1H), 2.47 (m, 2H), 2.09(m, 2H), 1.90 (m, 2H).

Example 3184-(cyclopropylamino)-2-(2,2-difluorobenzo[d][1,3]dioxol-5-ylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₁₅H₁₃N₅O₃F₂ as (M+H)⁺ 350.1.

Example 3194-(cyclopropylamino)-2-(2-methylbenzo[d]thiazol-5-ylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₁₆H₁₆N₆OS as (M+H)⁺ 341.0.

Example 3202-(benzo[d]thiazol-5-ylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₁₅H₁₄N₆OS as (M+H)⁺ 327.1.

Example 3214-(cyclopropylamino)-2-(imidazo[1,2-a]pyridin-6-ylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₁₅H₁₅N₇O as (M+H)⁺ 310.2.

Example 3222-(1H-benzo[d]imidazol-6-ylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₁₅H₁₅N₇O as (M+H)⁺ 310.1.

Example 3232-(1H-benzo[d][1,2,3]triazol-6-ylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₁₄H₁₄N₈O as (M+H)⁺ 311.2. ¹H NMR (CD₃OD,400 MHz): δ 8.63 (s, 1H), 8.42 (s, 1H), 7.92 (d, 1H), 7.58 (d, 1H), 3.04(m, 1H), 1.03 (m, 2H), 0.87 (m, 2H).

Example 3244-(cyclopropylamino)-2-(3-methyl-1H-indazol-6-ylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₁₆H₁₇N₇O₁ as (M+H)⁺ 324.

Example 3252-(benzo[c][1,2,5]thiadiazol-5-ylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine. MS found for C₁₄H₁₃N₇OS as (M+H)⁺ 328.2.

Example 3262-(7-chloro-1H-indazol-6-ylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine, after chlorinating the 6-aminoindazole ring usingN-chlorosuccinimide. MS found for C₁₅H₁₇N₇OCl as (M+H)⁺ 344.2, 346.2.

Example 3272-(3-chloro-1H-indazol-5-ylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Example 326. MS found for C₁₅H₁₇N₇OCl as (M+H)⁺ 344.2,346.2. ¹H NMR (CD₃OD, 400 MHz): δ 8.24 (s, 1H), 8.18 (s, 1H), 7.59 (m,2H), 2.97 (m, 1H), 0.80 (m, 2H), 0.64 (m, 2H).

Example 3282-(1-(2-amino-2-oxoethyl)-1H-indazol-6-ylamino)-4-(cyclopropylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1 using cyclopropylamine in place ofcyclobutylamine, using an intermediate derived from 6-nitroindazole andethyl bromoacetate. MS found for C₁₇H₁₈N₈O₂ as (M+H)⁺ 367.2.

Example 3294-(cyclobutylamino)-2-(2,2-difluorobenzo[d][1,3]dioxol-5-ylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1. MS found for C₁₆H₁₅N₅O₃F₂ as (M+H)⁺ 364.1.

Example 3304-(cyclobutylamino)-2-(imidazo[1,2-a]pyridin-6-ylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1. MS found for C₁₆H₁₇N₇O as (M+H)⁺ 324.2.

Example 3314-(cyclobutylamino)-2-(3-methyl-1H-indazol-6-ylamino)pyrimidine-5-carboxamide

The above compound was prepared using a procedure similar to thatdescribed in Scheme 1. MS found for C₁₇H₁₉N₇O as (M+H)⁺ 338.0.

Example 332

This example illustrates methods for evaluating the compounds of theinvention, along with results obtained for such assays. The in vitro andin vivo human syk activities of the inventive compounds can bedetermined by various procedures known in the art, such as a test fortheir ability to inhibit the activity of human plasma syk. The potentaffinities for human syk inhibition exhibited by the inventive compoundscan be measured by an IC₅, value (in nM). The IC₅₀ value is theconcentration (in nM) of the compound required to provide 50% inhibitionof human syk proteolytic activity. The smaller the IC₅₀ value, the moreactive (potent) is a compound for inhibiting syk activity.

An in vitro assay for detecting and measuring inhibition activityagainst syk is as follows:

Inhibition of Syk Tyrosine Phosphorylation Activity

Potency of candidate molecules for inhibiting syk tyrosinephosphorylation activity is assessed by measuring the ability of a testcompound to inhibit syk-mediated tyrosine phosphorylation of asyk-specific substrate.

SYK tyrosine phosphorylation activity is measured using the LANCE™Technology developed by Perkin Elmer Life and Analytical Sciences(Boston, Mass.). LANCE™ refers to homogeneous time resolved fluorometryapplications using techniques such as time-resolved fluorescenceresonance energy transfer assay (TR-FRET) (see generally for proceduresin Perkin Elmer Application Note—How to Optimize a Tyrosine Kinase AssayUsing Time Resolved Fluorescence-Based LANCE Detection,wwww.perkinelmer.com/lifesciences). The assay principle involvesdetection of a phosphorylated substrate using energy transfer from aphosphospecific europium-labeled antibody tostreptavidin-allophycocyanin as an acceptor.

To test the ability of candidate molecules to inhibit SYK tyrosinephosphorylation activity, molecules are reconstituted in 30% DMSO andserially diluted 1:3 with the final dilution containing DMSO in theabsence of the candidate molecule. The final DMSO concentration in theassay is 3%. Kinase assays are performed as a two part reaction. Thefirst reaction is a kinase reaction and which comprises of a candidatemolecule, full length active recombinant SYK enzyme (Millipore, Calif.)and biotin-labeled SYK-specific substrate biotin-DEEDYESP-OH. The secondreaction involves termination of the kinase reaction and thesimultaneous addition of the detection reagents-europium-labeledanti-phosphotyrosine reagent (Eu-W1024-PY100, Perkin Elmer, Boston,Mass.) and Streptavidin-Allophycocyanin detection reagent (SA-APC,Prozyme, Calif.). The kinase reaction is performed in a black U-bottom96-well microtitre plate. The final reaction volume is 50 μL andcontains a final concentration of 1 nM active SYK enzyme, 550 nMSYK-substrate, and 100 μM ATP diluted in a buffer containing 50 mM TrispH 7.5, 5 mM MgCl₂, and 1 mM DTT. The reaction is allowed to proceed for1 hour at room temperature. The quench buffer contains 100 mM Tris pH7.5, 300 mM NaCl₂, 20 mM EDTA, 0.02% Brij35, and 0.5% BSA. The detectionreagents are added to the reaction mixture at the followingdilutions-1:500 for Eu-W1024-PY100 and 1:250 for SA-APC. The kinasereaction is terminated by the addition of 50 μL quench buffer containingthe detection reagents. The detection is allowed to proceed for 1 hr atroom temperature. Detection of the phosphorolated substrate in theabsence and presence of inhibitors is measured in the TR-FRETinstrument, Analyst HT (Molecular Probes, Sunnyvale, Calif.) and thecondition for measurements are set up using CriterionHost Release 2.0(Molecular Probes, Sunnyvale, Calif.). The settings used are a follows:excitation 360 nm, emission 665-7.5 nm, beam splitter 350 nm 50/50,flash 100 pulses, delay 60 us, integration 400 us, z-height 2 mm.Inhibition of SYK-tyrosine kinase activity is calculated as the maximumresponse observed in the presence of inhibitor, compared to that in theabsence of inhibitor. IC₅₀s were derived by non-linear regressionanalysis.

Intracellular phospho-flow cytometry was used to test compoundinhibition of Syk activity in intact non-Hodgkin's lymphoma cell linesRamos and SUDHL-6. 10×10⁶ cells in log phase growth were aliqoted; Sykkinase is activated by incubating cells for 10 minutes with 3 μg/mlantibody specific to the B cell receptor. Directly following, cells arefixed in 1% paraformaldehyde for 5 minutes at room temperature, washedin phosphate buffered saline, and then permeabilized by incubation for 2hours in ice cold methanol. Cells are again washed in phosphate bufferedsaline, then incubated for 30 minutes with antibody specific forphosphorylated Erk (Y204) and BLNK (Y84), which are indicators of Sykkinase activity, and phosphorylated Syk (Y352), a measure of Src familykinase activity. All antibodies used are purchased from BD Pharmingen(San Jose, Calif.). After incubation with antibodies, cells are againwashed and subjected to flow cytometry. Representative data detailinginhibition of B cell receptor signaling by compounds are shown in Table1 as IC₅₀ ranges.

The anti-proliferative effects of compounds on non-Hodgkin's lymphoma Bcell lines SUDHL-4, SUDHL-6, and Toledo was also assessed. SUDHL-4 andSUDHL-6 require B cell receptor signaling for growth and survival, whilethe Toledo cell line (serving here as a negative control) does not.Cells were aliquoted into each well of a 96-well plate and incubatedwith increasing concentrations of compound for 72 hours, after whichcell survival and proliferation was determined using the MTT assay(Chemicon International, Inc., Temecula, Calif.) following protocolssupplied by the manufacturer. Data are detailed in Table 2 as IC₅₀values plus or minus standard deviations from 5 or 6 independentexperiments.

Induction of apoptosis in non-Hodgkin's lymphoma B cell lines SUDHL-4,SUDHL-6, and Toledo was assessed by measuring the apoptosis markerCaspase 3. Cells were incubated with 1, 3, or 10 μM compound for 24, 48,and 72 hours. At the conclusion of each time point, cells were processedfor flow cytometry analysis using the Monoclonal Rabbit Anti-ActiveCaspase-3 Antibody Kit and related protocols (BD Pharmingen). Data fromtwo independent experiments are presented in Tables 3A and 3B,representing the percent of total cells undergoing apoptosis followingincubation with compounds under the indicated conditions.

Syk activity is not only required for B cell signaling, proliferation,and survival, as shown, but is also critical for cellular activationupon cross-linking of the B cell receptor. B cell activation leads toincreased cell surface expression of several proteins involved in cellsignaling, antigen presentation, and adhesion. Among these, CD80, CD86,and CD69 are commonly measured to determine B cell activation status.Therefore, primary mouse B cells isolated from spleen were aliquoted andincubated with increasing concentrations of compound (0.05 to 2 μM) inthe presence of goat anti-mouse IgD (eBiosciences, Inc., San Diego,Calif.) for 20 hours to cross-link the B cell receptor. Following, cellswere washed and incubated for 30 minutes on ice with antibodies specificfor the CD80, CD86, and CD69 B cell activation markers. B cells wereidentified from the pooled population by staining with the B cell markerCD45RO. All antibodies were purchased from BD Pharmingen. Table 4depicts the IC₅₀ range in which these compounds inhibited B cellreceptor induced activation of mouse primary B cells.

In the table below, activity in the Syk and/or Jak assays is provided asfollows: +++++=IC₅₀<0.0010 μM; ++++=0.0010 μM<IC₅₀<0.010 μM, +++=0.010μM<IC₅₀<0.10 μM, ++=010 μM<IC₅₀<1 μM, +=IC₅₀>1 μM.

TABLE 7 Example UV MW MH+ Syk IC50 1 409.49 410.0 +++ 410.2 2 461.53462.3 ++ 3 395.47 396.0 +++ 396.3 396.4 ES (+) MS M + H = 396 4 423.52424 ++ 5 411.51 412.3 ++ 6 445.53 446.1 ++ 7 397.48 398.3 ++ 8 411.51412 ++ 9 413.48 413 ++ 10 427.51 428 ++ 12 409.49 410.2 ++ 13 393.45394.2 ++ 14 369.43 370.2 ++ 15 383.46 384.2 ++ Turb Spray MS [M + 1] =384 Turbo Spray MS [M + 1] = 384 16 437.43 438.2 ++ 17 459.55 M + 1 =460 ++ 18 445.53 446 ++ 32 557.66 558.2 ++ 33 471.53 472.2 ++ 34 429.91430.0, 432.0 +++ 35 443.94 444.0, 446.0 +++ 36 256, 409.49 410.3 +++249.5 420.2 420.4 ES (+) MS [M + 1] = 410 Turbo Spray MS [M + 1] = 41037 421.51 422.4 +++ 38 425.49 426.2 +++ ES (+) MS [M + 1] = 426 TurboSpray MS [M + 1] = 426 39 409.45 410.2 ++ 40 413.46 414.2 +++ 41 452.56453.45 ++++ 42 452.56 453.41 +++ 45 466.59 467.46 ++ 46 495.59 496.45 ++48 452.56 453.45 +++ 49 495.59 497 ++ 50 441.54 ES (+) MS [M + 1] = 442++ 51 413.48 414.4 ++ 52 413.48 414.4 ++ 53 394.44 395.2 ++ 54 413.48414.3 ++ 55 438.49 439.4 +++ 56 409.49 410.4 +++ 58 426.48 427.4 ++ 59440.51 441.3 ++ 60 408.47 409.3 ++ 63 466.55 467.4 ++ 64 471.57 472.3 ++65 413.48 414.3 ++ 66 427.51 428.4 ++ 67 452.52 453.3 +++ 69 429.48430.3 ++ 70 466.55 467.4 +++ 71 425.49 426.3 +++ 72 207.8, 500.56 501.3++ 293.8 73 228.5, 410.53 411.43 +++ 77 410.48 342 +++ 78 438.54 439.3++ 86 219.2 498.59 499.4 ++ 87 208.6 512.62 513.4 ++ 92 317.78 318.0,320.0 ++ 93 396.46 397 ++ 96 428.52 429.3 (M + 1) +++ 97 430.53 431.3(M + 1) +++ 98 459.57 460.2 +++ 99 431.52 432.0 +++ 432.2 ES (+) MS [M +1] = 432 ES (+) MS [M + H] = 432 Turb Spray MS [M + 1] = 432 Turbo SprayMS [M + 1] = 432 100 445.55   426 (M + H) +++ 446 446.2 446.2 (M + H)446.4 ES (+) MS [M + 1] = 446 Turbo Spray MS [M + 1] = 446 101 203.6,445.55 446.0 +++ 230.8, ES (+) MS 258.0 [M + 1] = 446 103 362.41 363 +++104 370.41 371 +++ 105 398.47 399 ++ 106 390.47 391 +++ 107 356.39 355+++ 108 201.4, 384.44 385.0 ++ 275.8 ES (+) MS [M + 1] = 385 109 348.39349.0 +++ 349.1 110 341.38 342 ++ 111 331.4 332.1 ++ 112 423.48 424.1(M + 1) ++ 113 394.48 395.3 (M + 1) +++ 114 449.56 450.3 ++ 115 463.59464.3 ++ 116 465.56 466.3 ++ 117 457.56 458.2 +++ 118 409.49 410.2 +++119 409.49 410.2 ++ 120 362.39 363.2 ++ 121 402.48 403.2 +++ 122 411.51M + 1 = 412 ++ 123 407.52 408.3 (M + 1) +++ 124 423.52 424.2 +++ 125402.48 M + 1 = 403 +++ 127 406.47 M + 1 = 407 +++ 128 392.44 M + 1 = 393+++ 129 361.42 362.1 ++ 130 367.46 368.3 +++ 131 430.52 MS: 431.42 (M +H) ++ 135 381.48 382.5 (M + 1) +++ 136 426.46 M + 1 = 427 ++ 137 431.5M + 1 = 432 +++ 138 429.46 M + 1 = 430 +++ 139 454.51 M + 1 = 455 +++140 355.4 356.3 ++ 141 367.33 368 ++ 168 297.36 298 ++ 169 326.36 327 ++170 380.45 381 +++ 171 207.3, 388.86 389.0, 391.0 ++ 266.2 ES (+) MS M +H = 389, Chlorine patteren 172 374.83 375.0, 377.0 ++ 173 366.43 367.1++ 174 397.48 398.3 +++ ES (+/−) MS M + H = 398 Turbo Spray MS [M + 1] =398 175 409.49 410 +++ 176 206.1, 423.52   424 (M + H) +++ 268.7   424(M + H) 424.0 424.4 ES (+) MS [M + 1] = 424 177 424.51 425.2 ++ 178388.86 389.0, 391.0 ++ 179 374.4 375 ++ 180 326.36 8 ++ 181 340.39 341++ 182 459.53 460.1 (M + 1) ++ 183 352.42 MS: 353.2 (M + H) ++ 184335.37 MS: 336.3 (M + H) ++ 185 337.35 MS: 338.2 ++ 186 351.43 MS: 352.2(M + H) ++ 187 328.33 329.2 +++ 188 346.39 MS: 347.3 (M + H) ++ 189392.46 393.3 (M + 1) +++ 191 382.47 383.3 ++ 192 346.39 MS: 347.3 (M +H) +++ 193 361.42 362.1 (M + 1) +++ 194 308.35 309.2 +++ 195 396.45397.2 +++ 196 445.55 446.2 +++ 197 363.43 MS: 364.2 (M + H) ++ 198232.0, 381.44 ES (+) MS +++ 305.0 [M + 1] = 382 199 352.4 M + 1 = 353+++ 200 366.43 M + ! = 367 +++ 201 252, 395.47 ES (+) MS [M + 1] = 396+++ 299.5, 313.6 202 232.0, 409.49 ES (+) MS [M + 1] = 410 ++ 252.1 203356.39 M + ! = 357 +++ 204 370.41 M + 1 = 371 +++ 205 409.49 M + 1 = 410+++ 206 423.52 M + 1 = 424 +++ 207 409.49 410.2 +++ 208 423.52 424.2 +++209 409.49 410.2 ++ ES (+) MS [M + 1] = 410 210 423.52 424.2 ++ 211321.34 322.1 ++ 212 367.41 368.1 +++ 212 411.47 412.5 ++ 213 432.5 m + 1= 433 +++ 214 366.43 367.1 ++ 215 380.45 M + 1 = 381 ++ 216 361.41 362.4(M + 1) +++ 218 205.3 409.49 ES (+) MS [M + 1] = 410 ++ 232.1 252.3 219395.47 396.5 ++ 220 397.44 398.5 ++ 222 326.4 327 +++ 223 340.39 341 ++225 323.36 324 +++ 226 313.36 314 +++ 227 354.46 355.2 ++ 228 283.34 284++ 229 368.44 369 +++ 230 354.41 355 ++ 231 340.39 341 +++ 233 327.34328.2, 329.1 ++ 234 205.0, 354.41 355.0 +++ 254.4 ES (+) MS M + H = 355Turbo Spray MS [M + 1] = 355 235 230.8, 352.44 353.2 +++ 280.5 ES (+) MS[M + 1] = 353 236 201.4, 368.44 369.0 +++ 271.0 ES (+) MS [M + 1] = 369237 370.43 371 +++ 238 201.4, 367.46 368.3 +++ 227.3 ES (+) MS [M + H] =368 Turbo Spray MS [M + 1 ] = 368 239 388.42 389 +++ 240 395.47 396.2+++ 241 396.46 397.2 +++ 242 388.42 389 +++ 244 347.4   348 (M + H) +++348.1 348.3   349 (M + H) ES (+) MS [M + 1] = 348 Turbo Spray MS [M + 1]= 348 245 315.4 316.1 ++ 246 445.55 446.3 (M + 1) ++ 247 353.43 354.2+++ 248 336.36 MS: 337.3 (M + H) ++ 249 308.35 309.2 +++ 250 309.33310.2 +++ 251 336.36 MS: 337.1 (M + H) ++ 252 309.33 310.2 +++ ES (+) MS[M + 1] = 310 ES (+) MS [M + H] = 310 Turbo Spray MS [M + 1] = 310 253320.36 321.2 +++ 254 395.47 M + 1 = 396 +++ 256 341.37 342.2, 343.2 ++257 366.47 367.3 ++ 258 382.47 383.3 +++ 259 384.46 385 +++ 260 368.44369 +++ 261 201.5, 381.48 382.3 ++ 228.5 ES (+) MS M + H = 382 262402.45 403 ++ 263 402.45 403 +++ 264 200.8, 381.44 ES (+) MS [M + 1] =382 +++ 231.8, 304.8 265 397.44 398.4 ++ 266 411.47 412.5 ++ 267 425.49426.5 ++ 268 423.52 424 +++a 269 210.8, 437.55 438.3 ++ 253.2 ES (+) MSM + H = 438 270 411.51 412.3 +++ 271 402.89 403.0, 405.0 +++ 272 388.42389 ++ 273 340.39 341 ++ 274 354.41 355.2 ++ 288 275 313.36 MW = 313.35;++ M + 1 = 314.2 289 277 361.41 MW = 361.4; ++ M + 1 = 362.2 290 265353.3 MW = 353.3; ++ M + 1 = 354.1 291 205, 362.39 MW = 362.4; ++ 267M + 1 = 363.2 292 290 386.43 MW = 386.4, M + 1 = 387.2 ++ 309 338.37339.0 (M + 1) +++ 314 368.4 369 ++ 320 326.38 327.1 ++ 322 309.33 310.1+++ 323 310.32 311.2 +++ 324 218.6 323.36 ES (+) MS [M + 1] = 324 +++247.8 325 327.37 328.2 ++ 326 343.78 344.2, 346.2 ++ 328 366.39 367.2 ++330 324.35 325 +++ 331 214.9 337.39 ES (+) MS [M + 1] = 338 +++ 252.1,298.2Inhibition of GPVI-Mediated Platelet Function In Vitro

The ability for candidate molecules to inhibit syk-mediated plateletfunctions are tested by measuring the inhibition the GPVI-specificagonist Convulxin-induced human platelet calcium-mobilization oraggregation. Calcium-mobilization is assessed in human washed plateletsin a 96-well microtiter format. Aggregation is assessed in a 96-wellmicrotiter assay (see generally the procedures in Jantzen, H. M. et al.(1999) Thromb. Hemost. 81:1111-117) or standard cuvette lighttransmittance aggregometry using human platelet-rich plasma (PRP).

Inhibition of Convulxin-Mediated Platelet Calcium-Mobilization In Vitro

Inhibition of Convulxin-induced calcium-mobilization was determined inhuman washed platelets using the FLIRP Calcium 3 Assay Kit (MolecularDevices, Sunnyvale, Calif.). For preparation of washed platelets, humanvenous blood is collected from healthy, drug-free volunteers into ACD(85 mM sodium citrate, 111 nM glucose, 71.4 mM citric acid) containingPGI₂ (1.25 ml ACD containing 0.2 μM PGI₂ final; PGI₂ was from Sigma, St.Louis, Mo.). Platelet-rich plasma (PRP) is prepared by centrifugation at160×g for 20 minutes at room temperature. Washed platelets are preparedby centrifuging PRP for 10 minutes at 730 g and resuspending theplatelet pellet in CGS (13 mM sodium citrate, 30 mM glucose, 120 mMNaCl; 2 ml CGS/10 ml original blood volume). After incubation at 37° C.for 15 minutes, the platelets are collected by centrifugation at 730 gfor 10 minutes and resuspended at a concentration of 3×10⁸ platelets/mlin Hepes-Tyrode's buffer (10 mM Hepes, 138 mM NaCl, 5.5 mM glucose, 2.9mM KCl, 12 mM NaHCO₃, pH 7.4). This platelet suspension is kept >45minutes at room temperature before use in calcium mobilization assays.

For 96-well plate Calcium-mobilization experiments, equal volumes of3×10⁸ washed platelets/ml were incubated with equal volumes of Calcium-3Assay Reagent A resuspended in 1× Hank's Balanced Salt Solution, pH 7.4,20 mM Hepes buffer. The total reaction volume of 0.2 ml/well includes1.5×10⁸/ml washed platelet/Calcium-3 Assay reagent A mix, 10 μMEptifibatide (Millennium Pharmaceuticals Inc, Cambridge, Mass.), serialdilutions (1:3) of test compounds in 0.75% DMSO. DMSO alone is added to1 well of each 8 set to allow for a maximal calcium-mobilizationreading. After 20 minutes preincubation at room temperature the 96-wellmicroplate reader is loaded into the FlexStation (Molecular Devices,Sunnyvale, Calif.). The FlexStation experimental conditions formeasuring Calcium mobilization are set up using SOFTMax Pro. Thesettings used are detailed below. Fluorescence parameters-assay mode:flex, excitation 485 nM, 525 nM with a cut-off of 515 nM; Parameters-PMTsensitivity-6, pipette height 230 μl, read time 2 minutes and 40seconds, read intervals 2 seconds, temperature-23-25° C. After 18seconds of baseline reading, calcium-mobilization is initiated by theaddition of Convulxin to a final concentration of 125 ng/ml. Inhibitionof calcium-mobilization was calculated as the maximum response observedin the presence of inhibitor, compared to that in the absence ofinhibitor. IC₅₀s were derived by non-linear regression analysis.

Inhibition of Convulxin-Mediated Platelet Aggregation In Vitro

For preparation of human platelet-rich plasma for aggregation assays,human venous blood was collected from healthy, drug-free volunteers into0.38% sodium citrate (0.013 M, pH 7.0 final). Platelet-rich plasma (PRP)is prepared by centrifugation of whole blood at 160×g for 20 minutes atroom temperature. The PRP layer is removed, transferred to a new tube,and the platelet count is adjusted, if advantageous, to achieve aplatelet concentration of ˜3×10⁸ platelets/ml using platelet-poor plasma(PPP). PPP is prepared by centrifugation of the remaining blood sample(after removal of PRP) for 20 minutes at 800×g. This preparation of PRPcan subsequently be used for aggregation assays in either a 96-wellplate or standard cuvette aggregometry.

Inhibition of Convulxin-induced aggregation is determined in 96-wellflat-bottom microtiter plates using a microtiter plate shaker and platereader similar to the procedure described by Frantantoni et al., Am. J.Clin. Pathol. 94, 613 (1990). All steps are performed at roomtemperature. For 96-well plate aggregation using platelet-rich plasma(PRP), the total reaction volume of 0.2 ml/well includes 190 μl of PRP(˜3×10⁸ platelets/ml, see above), and 5 μl of either serial dilution oftest compounds in 30% DMSO or buffer (for control wells). After 20minutes preincubation at room temperature 5 μl of 320 ng/ml Convulxinagonist solution is added to each well to give a final concentration of8 ng/ml Convulxin. The plates are then agitated for 5 min on amicrotiter plate shaker and the 5 minute reading is obtained in themicrotitre plate reader (Softmax, Molecular Devices, Menlo Park,Calif.). Aggregation is calculated from the decrease of OD at 650 nm att=5 minutes. IC₅₀s were derived by non-linear regression analysis.

Inhibition of Convulxin-induced aggregation was also determined forcuvette light transmittance aggregation assays, serial dilutions (1:2)of test compounds were prepared in 30% DMSO in a 96 well V-bottom plate(final DMSO concentration in the cuvette was 0.3%). The test compound (5μl of serial dilutions in DMSO) was preincubated with PRP for 20 minutesprior to initiation of aggregation reactions, which is performed in aChronoLog aggregometer by addition of agonist (125-250 ng/ml Convulxin)to 495 μL of PRP at 37° C. The aggregation reaction is recorded for 4min, and maximum extent of aggregation is determined by the differencein extent of aggregation at baseline, compared to the maximumaggregation that occurs during the 4 minute period of the assay.Inhibition of aggregation was calculated as the maximum aggregationobserved in the presence of inhibitor, compared to that in the absenceof inhibitor. IC₅₀s were derived by non-linear regression analysis.

Examples of compounds and their syk and PRP IC₅₀ values are given intables 1-5.

Calcium Flux Assay in Ramos Cells Induced by BCR Cross-Linking

Ramos cells (2G6.4C10, Burkitt's lymphoma, ATCC Item Number: CRL-1923)are sub-cultured at 5×10⁵ cells/ml in fresh medium 3 or 4 days ahead ofexperiments. Cells are harvest and re-suspend in fresh medium at 8×10⁶cells/ml before dye-loading. An equal volume of Calcium 3 loading dye(Molecular Device) is added and mixed into cell suspension. Loadingcells are dispensed in a 96 well plate and incubated 30 min. Compoundsare then added in the dye-loaded cells and incubated for another 30 min.Spin cell down at 1000 rpm for 3 min before fluorescence measurement inFlexStation. BCR stimulation is carried by the addition of 5 μg/mlantibody (AffiniPure F(ab′)₂ fragment Donkey anti-human IgM, JacksonImmunoResearch Laboratories).

Calcium Flux Assay in Jurkat Cells Induced by TCR Cross-Linking

The protocol is very similar to B cell calcium flux as described in theprevious section. The only differences are that T cells (clone E6-1,Acute T cell Leukemia, ATCC Item Number: Tib-152) and anti-human CD3(Functional Grade Purified anti-human CD3, clone OKT3, eBioscience, No.16-0037) replaced B cells and anti-human IgM. Cell density is kept thesame but antibody is used at a concentration of 100 ng/ml.

IL-2 Secretion in Jurkat Cells Induced by TCR Cross-Linking

Jurkat cell propagation and compound incubation procedures are the sameas described in Jurkat calcium flux assay in the previous section.Antibody (anti CD3, OKT3) is coated onto a fresh plate (without cells)at 100 ng/well. Cells are suspended at 8×10⁶ cells/ml and incubated withcompounds for 30 min in a separate plate. At the end of incubation,cells are transferred to the antibody-coated plate and incubated for 16hours. 100 μl of cell medium after incubation is used for IL-2measurement after incubation. IL-2 level is determined using an IL-2ELISA kit (Human IL-2 ELISA kit II, BD Bioscience, No. 550611).

Example 333 Millipore Upstate KinaseProfiler™ Screening

This assay is a direct measurement of the effect of compound on thecatalytic activity of JAK3. Purified human JAK3 (GenBank AF513860)sequence (residue 781—C terminus) was obtained from insect cells. Thecatalytic hydrolysis of ATP is measured using a radiometric filterbinding method. Incubation of kinase with ³³[P]ATP and substrate leadsto incorporation of ³³[P] into the substrate which can then be separatedfrom the other reaction components by filtration. Assays were performedusing 10 μM ATP and in the absence or presence of 1, 0.3, or 0.1 μMcompound. Activity was expressed as % of inhibition of control.

TABLE 8 Inhibition (%) of catalytic activity of JAK3 by 1, 0.3 or 0.1 μMcompound as determined by Millipore using their KinaseProfiler Assay.Concentration (μM) Compound 1 μM 0.3 μM 0.1 μM

98 96 97

100 84 ND ND: not done

Example 334 Ambit KinomeScan Screening

This assay is an ATP-site dependent competition binding assay in whichhuman kinases of interest are fused to a proprietary tag (T7bacteriophage). The amount of kinase bound to an immobilized,active-site directed ligand is measured in the presence and absence ofthe test compound. Ambit's JAK assays use kinase domains and notfull-length proteins. The domain used for JAK1 binding is the pseudokinase domain while that for JAK3 binding is the catalytic domain (MazenW Karaman, Sanna Herrgard, Daniel K Treiber, et. al. A Quantitativeanalysis of kinase inhibitor selectivity. Nature Biotechnology, 2008,Volume 26, No. 1, Page 127-132).

TABLE 9a KD values (nM) for compound binding inhibition of JAK1 and JAK3to immobilized ligand in the Ambit KinomeScan assay. Compound JAK1 JAK3

6.7 2.9

11 3

4.7 2.7

14 4.1

TABLE 9b Potency and Specificity of Kinase Inhibtion (IC50 in nM)Compound Syk Jak 1 Jak 2 Jak 3 EXAMPLE 99 15 6.7 3.2 0.8 P420-89 31 6.22.0 0.6

TABLE 10 (Ambit Panel) inhibition of Kinases in Kd (nM)

JAK1 6.7 4.7 (Kin. Dom. 1) JAK2 5.1 5.1 (Kin. Dom. 2) JAK3 2.9 2.7 (Kin.Dom. 2) Syk 98 4.5

JAK1 14 (Kin. Dom. 1) JAK2 10 (Kin. Dom. 2) JAK3 4.1 (Kin. Dom. 2) Syk9.4

Example 335 JAK3/STAT6 Cellular Assay

Stimulation of Ramos B cells by interleukin 4 (IL4) leads to signalingthrough JAK1/JAK3 resulting in phosphorylation of STAT6 (signaltransducers and activators of transcription). The effect of compounds oninhibition of JAK3 and/or JAK1 can be assessed by measuring the amountof phosphorylated STAT6. This is performed by Western blotting using aspecific phospho-STAT6 antibody.

Ramos B cells were suspended in 10 mM Hepes-buffered RPMI media (2×10⁷cells/ml). Cells (90 μl) were incubated with 10 μl 3.3 μg/ml interleukin4 (R & D Systems Inc, cat #204-IL; final concentration: 0.33 μg/ml).Incubations were for 10 min at 37° C. in the absence or presence of 2 μlcompound diluted in 30% DMSO. Reactions were terminated by the additionof an equal volume of 2× lysis buffer (100 mM TRIS-HCl pH 8.0, 2%Triton-X-100, 5 mM EDTA, 250 mM NaCl, 20% glycerol, 1.25 mM PMSF, 5 mMsodium orthovandate, 5 mM β-glycerophosphate, mini complete EDTAprotease inhibitor cocktail (Sigma)).

Samples were incubated with 1 μl of the nuclease, benzonase (Novagen,cat #71205-3) for 1 hour, room temperature and then 50 μl 5× loadingbuffer (330 mM TRIS pH 6.8, 9.5% SDS, 34% glycerol, 0.01% bromophenolblue, 10% beta-mercaptoethanol) was added.

Cell lysates (15 μL) were subjected to SDS-PAGE (Novex 4-12%TRIS-glycine gels, Invitrogen) under reducing conditions, followed byelectroblot-transfer onto nitrocellulose membranes. Membranes were thenincubated in Zymed blocking buffer (Invitrogen) for 1 hr at roomtemperature (RT) then overnight at 4° C. with 1:500 antiphosphotyrosine—STAT6 (Cell Signaling Technology, cat #9364) primaryantibody in Zymed blocking buffer. Following 5×10 min washes withTris-buffered saline, 0.25% NP40 (TBSN), blots were incubated for 1 hrat room temperature in the presence of 1:10,000 HRP-conjugated donkeyanti-rabbit secondary antibody (Amersham Biosciences, cat #NA934V) inZymed blocking buffer. After 4×10 min TBSN washes, blots were visualizedby ECL (Pierce Western Lightening, Perkin Elmer cat #NEL101). In orderto determine total β3 content, blots were stripped, washed 4× with TBSN,and re-probed with 1:2000 C3A antibody in block buffer overnight at 4°C. After 4×10 min TBSN washes, blots were incubated with 1:10,000 goatanti-mouse secondary antibody in blocking buffer, washed 4 more timeswith TBSN and exposed to Western Lightening reagent. Levels ofstimulation over background and the extent of inhibition of compound aredetermined by densitometry.

Example 336 Inhibition of JAK Kinase Activity Assay for Ramos B-CellLine Stimulated with IL-4

These examples illustrate methods for evaluating the in vitro and invivo human JAK kinase activities of the inventive compounds can bedetermined by various procedures known in the art, such as a test fortheir ability to inhibit the activity of human plasma JAK kinase. Thepotent affinities for human JAK kinase inhibition exhibited by theinventive compounds can be measured by an IC₅, value (in nM). The IC₅₀value is the concentration (in nM) of the compound required to provide50% inhibition of human JAK kinase activity. The smaller the IC₅₀ value,the more active (potent) is a compound for inhibiting JAK kinaseactivity.

An in vitro assay for detecting and measuring inhibition activityagainst JAK kinase is as follows:

The activity of the compounds for JAK kinases is confirmed in cellularassays designed to test for JAK inhibition. Briefly, JAK inhibition istested in human Ramos B-cells activated with cytokine Interleukin-4(IL-4). Twenty to 24 hours post stimulation, the cells are stained forupregulation of CD23 and analyzed by FACS. Stimulation of the B-cellswith IL-4 leads to the activation of the JAK/STAT pathway throughphosphorylation of the JAK kinase JAK1 and JAK3, which in turnphosphorylate and activate transcription of factors STAT-5 and STAT-6.The low-affinity IgE receptor (CD23) is upregulated by activated STAT-5.

For the assay, human Ramos B-cells (ATCC, Catalog No. CRL-1596) arecultured in RPMI 1640 medium (Cellgro, Catalog No. 10-040-CM) containing10% fetal bovine serum (JRH, Catalog No. 12106-500M) according to thepropagation protocol supplied with the cells, and maintained at adensity of approximately 3.5×10⁵ cells/ml. The day before the assay, thecells are diluted to 3.5×10⁵ cells/ml to insure they are in thelogarithmic growth phase. The cells are spun down, and suspended in RPMI1640 medium (Cellgro, MediaTech, Inc., Herndon, Va., Cat No. 10-040-CM)containing 5-10% fetal bovine serum (FBS), heat inactivated (JRHBiosciences, Inc, Lenexa, Kans., Cat No. 12106-500M) according to ATCCpropagation protocol. Cells are maintained at a density of 3.5×10⁴⁻⁵cells/ml. The day before the experiment, Ramos B-cells are diluted to3.5×10⁵ cells/mL to ensure that they are in a logarithmic growth phaseand aliquots dispensed into a 96-well tissue culture plate. Cells areincubated with test compound (dissolved in DMSO) or DMSO (control) for 1hr at 37° C. and then stimulated with IL-4 (Pepotech, Catalog No.200-04) for 20-24 hours (final concentration is 50 Units/ml).

Cells are spun down and suspended in RPMI with 5% serum. 5×10⁴ cells areused per point in a 96-well tissue culture plate. Cells arepre-incubated with compound or DMSO (Sigma-Aldrich, St. Louis, Mo., CatNo. D2650) vehicle control for 1 hour in a 37° C. incubator. Cells arethen stimulated with IL-4 (Peprotech Inc., Rocky Hill, N.J., Cat No.200-04) for a final concentration of 50 units/mL for 20-24 hours. Cellsare then spun down and stained with anti-CD23-PE (BD Pharmingen, SanDiego, Calif., Cat No. 555711) and analyzed by FACS. Detection isperformed using a BD LSR I System Flow Cytometer, purchased from BectonDickinson Biosciences of San Jose, Calif.\

Proliferation is measured using CellTiter-Glo® Luminescent CellViability Assay (Promega), which determines the number of viable cellsin culture based on quantitation of the ATP present, as an indicator ofmetabolically active cells. The substrate is thawed and allowed to cometo room temperature. After mixing the Cell Titer-Glo reagent and diluenttogether, 100 μL is added to each well. The plates are mixed on anorbital shaker for two minutes to induce lysis and incubated at roomtemperature for an additional ten minutes to allow the signal toequilibrate. Detection is performed using a Wallac Victor2 1420multilabel counter purchased from Perkin Elmer, Shelton, Conn.

On day two, A549 cells are pre-incubated with a 2,4-pyrimidinediaminetest compound or DMSO (control) (Sigma-Aldrich, St. Louis, Mo., CatalogNo. D2650) for 1 hour. The cells are then stimulated with IFNγ (75ng/mL) (Peprotech Inc., Rocky Hill, N.J., Cat. No. 300-02) and allowedto incubate for 24 hours. The final test compound dose range is 30 μM to14 nM in 200 μL F12K media containing 5% FBS, 0.3% DMSO.

On day three, the cell media is removed and the cells are washed with200 μL PBS (phosphate buffered saline). Each well is trypsinized todissociate the cells, then neutralized by addition of 200 μL completeF12K media. Cells are pelleted and stained with an APC conjugated mouseanti-human ICAM-1 (CD54) (BD Pharmingen, San Diego, Calif., Catalog#559771) antibody for 20 minutes at 4° C. Cells are washed with ice coldFACS buffer (PBS+2% FBS) and surface ICAM-1 expression is analyzed byflow cytometry. Detection is performed using a BD LSR I System FlowCytometer, purchased from BD Biosciences of San Jose, Calif. Events aregated for live scatter and the geometric mean is calculated(Becton-Dickinson CellQuest software version 3.3, Franklin Lakes, N.J.).Geometric means are plotted against the compound concentration togenerate a dose response curve.

Example 337 Inhibition of Syk-Mediated Signal Transduction Through the BCell Receptor in Non-Hodgkin's Lymphoma Cell Lines

Cells were pre-treated for 1 hour without or with compound (0.02 to 2uM) prior to stimulation of B cell receptor singling by incubation ofcells with 3 μg/ml anti-mu antibody for 10 minutes at 37° C. Ca²⁺ fluxwas measured using the Calcium 3 loading dye and the FlexStation(Molecular Device). B cell receptor signaling was assayed byintracellular phospho-Flow Cytometry, following protocols supplied by BDPharmingen (San Jose, Calif.). Syk activation was measured by inductionof BLNK tyrosine phosphorylation at amino acid position 84 (pBLNK Y84)and induction of ERK1/2 tyrosine phosphorylation at amino acid position204 (pERK Y204). Activation of the Src family member Lyn was measured byinduction of Syk tyrosine phosphorylation at amino acid position 352(psyk Y352). Data are presented as μM IC₅₀s. Each compound effectivelyinhibited B cell receptor-induced Ca²⁺ fluxing and activation of Syk,but not the Src family member Lyn.

Example 338 Syk Inhibition Exerts an Anti-Proliferative Effect onNon-Hodgkin's Lymphoma Cell Lines

Cells were incubated with increasing concentrations of each compound,then evaluated at 72 hours for extent of proliferation using the MTTassay (company, city, state) following the manufacturer suppliedprotocol. Data are presented as μM IC50 values, representing the meanplus/minus standard deviation from 5 or 6 independent experiments. Eachcompound inhibited proliferation of SUDHL-4 and -6 cell lines, whichrely on Syk for survival and growth signals, in the low μM range. Toledocells which do not require Syk, was noticeably less sensitive to theanti-proliferative effects of Syk inhibition.

Example 339 Syk Inhibition Induces Apoptosis in Non-Hodgkin's LymphomaCell Lines

Data represent two independent experiments to evaluate the effect of Sykand Syk/JAK inhibition on survival of diffuse large non-Hodgkin'slymphoma B cell lines. SUDHL-4 and SUDHL-6 cells lines rely onSyk-mediated B cell receptor signaling for survival, while Toledo cellsdo not. Cells were incubated with compounds at the indicatedconcentrations and times; induction of apoptosis was measured by flowcytometry using the Caspase 3 Detection Kit (Sigma-Aldrich, Saint Luis,Mo.). Data are presented as the percent of total cells positive for theapoptosis marker, caspase 3. As expected, Syk inhibition resulted in theinduction of apoptosis in SUDHL-4 and -6 cell lines, but not the Toledocell line.

Example 340 Inhibition of Mouse Primary B Cell Activation by SykInhibitors

Mouse primary splenocytes were pre-treated for 1 hour with increasingconcentrations of each compound (0.05-2 μM) prior to addition of controlor goat anti-mouse IgD serum. Anti-IgD induced B cell activation wasmeasured 16 hours later by flow cytometry, staining for the activationmarkers CD80/86 and CD69.

Example 341 Mouse Model of Immune-Mediated Thrombocytopenia

Immune-mediated thrombocytopenia is caused by antibodies directedagainst platelet surface glycoproteins, antibodies againstdrug-containing complexes on the platelet surface, or by antibody-coatedcells or immune complexes that interact with the platelet surface.Select compounds were evaluated for their ability to inhibit plateletclearance in a mouse model of antibody-mediated thrombocytopenia. Inthis model, a rapid clearance of circulating platelets (approximately50%) results from the intravenous administration of a rat anti-mouseGPIIb (clone MWReg30) antibody (BD Biosciences, Pharmingen). To evaluatecapacity for inhibition of platelet clearance, compounds were suspendedinto 0.5% methycellulose in water and administered via oral gavage (100ul/mouse) at a time prior to antibody injection when the compound wouldachieve maximum plasma concentration (typically 1-2 hours based onseparate pharmacokinetic experiments for individual compounds). At 4 and8 hours after injection of antibody, terminal blood samples wereobtained from groups of vehicle and test article treated mice (n=5-10mice/group) via cardiac puncture. Blood was anticoagulated usingtrisodium citrate or EDTA. Whole blood samples were measured forplatelet counts on a hematology analyzer (Hemavet, Drew Scientific).Remaining blood was processed for plasma and compound concentrationsmeasured by mass spectrometry.

Platelet clearance was determined by measuring the difference inplatelet number between the average non-antibody treatment group andanimals administered the rat anti-mouse GPIIb antibody. Inhibition ofplatelet clearance was determined by comparing the difference betweenplatelet clearance of vehicle and compound treated animals.

Example 342 Mouse Model of Collagen Antibody Induced Arthritis

The inhibitory activity of select compounds was investigated in a mousemodel of collagen antibody induced arthritis (CAIA). Collagen inducedarthritis is mediated by autoantibodies to type II collagen andcomplement, thus arthritis can be induced by administration ofpolyclonal antibodies or a mixture of monoclonal antibodies to type IIcollagen. The CAIA model (Chondrex, Inc., Redmond, Wash.) uses a mixtureof 4 clones which recognize individual epitopes clustered within an 83amino acid peptide fragment of type II collagen. These epitopes sharecommon amino acid sequences with many different species of type IIcollagen including chicken, mouse, rat, bovine, porcine, monkey andhuman. The model utilizes a monoclonal antibody cocktail followed bybacterial lipopolysaccharide (LPS) to induce a severe and consistentarthritis in mice within 7 days. This model was developed based on thehypothesis that bacterial toxins absorbed through the gastrointestinaltract play a synergistic and pathologic role with autoantibodies to typeII collagen in triggering arthritis in patients with RheumatoidArthritis.

For these experiments, the monoclonal antibody cocktail (Lot # OC-708)was injected intravenously via tail vein at a dose of 4 mg/mouse (40mg/ml) on day 0 followed by intraperitoneal injection of LPS dilutedinto normal saline at a dose of 25 ug/mouse in 8 week old, female Balb/Cmice (Charles River, Inc.). Dosing of test articles was started justbefore or after the IV injection of antibody cocktail. Compounds weresuspended into 0.5% methylcellulose in water and administered via oralgavage (100 ul/mouse) daily for the duration of the 7-10 day study.Clinical inflammation scores were obtained daily. Inhibition of clinicalinflammation scores was determined based on the difference betweenvehicle and test article treated mice at the end of the experiment.Plasma concentrations represent peak concentration at 1 hour post lastdose on the day of study termination.

Example 343 Inhibition of IL-4 Induced JAK1/3 to Stat-6 Phosphorylationin Ramos B Cells

Ramos B cells were pre-treated for 1 hour with increasing concentrationsof compound, as indicated prior to addition of IL-4. Cells wereincubated with IL-4 for 10 minutes, and then subjected to intracellularflow cytometry to measure the percent inhibition of IL-4 induced Stat-6.

Example 344 Inhibition of IL-4 Induced JAK1/3 to Stat-6 Phosphorylationin Ramos B Cells

Ramos B cells were pre-treated for 1 hour with increasing concentrationsof compound, as indicated prior to addition of IL-4. Cells wereincubated with IL-4 for 10 minutes, and then subjected to intracellularflow cytometry to measure the percent inhibition of IL-4 induced Stat-6.

Example 345 Primary Human T-Cell Proliferation Assay Stimulated withIL-2

Primary human T-cells derived from peripheral blood and pre-activatedthrough stimulation of the T-cell receptor and CD28 proliferate in vitroin response to the cytokine Interleukin-2 (IL-2). This proliferativeresponse is dependent on the activation of JAK-1 and JAK-3 tyrosinekinases, which phosphorylate and activate the transcription factorStat-5.

Human primary T cells are prepared as follows. Whole blood is obtainedfrom a healthy volunteer, mixed 1:1 with PBS, layered on to FicollHypaque (Amersham Pharmacia Biotech, Piscataway, N.J., Catalog#17-1440-03) in 2:1 blood/PBS:ficoll ratio and centrifuged for 30 min at4° C. at 1750 rpm. The lymphocytes at the serum: ficoll interface arerecovered and washed twice with 5 volumes of PBS. The cells areresuspended in Yssel's medium (Gemini Bio-products, Woodland, Calif.,Catalog #400-103) containing 40 U/mL recombinant IL2 (R and D Systems,Minneapolis, Minn., Catalog #202-IL (20 μg)) and seeded into a flaskpre-coated with 1 μg/mL anti-CD3 (BD Pharmingen, San Diego, Calif.,Catalog #555336) and 5 μg/mL anti-CD28 (Immunotech, Beckman Coulter ofBrea Calif., Catalog #IM1376). The primary T-cells are stimulated for 3to 4 days, then transferred to a fresh flask and maintained in RPMI with10% FBS and 40 U/mL IL-2.

Primary T-cells are washed twice with PBS to remove the IL-2 andresuspended in Yssel's medium at 2×10⁶ cells/mL. 50 μL of cellsuspension containing 80 U/mL IL-2 is added to each well of a flatbottom 96 well black plate. For the unstimulated control, IL-2 isomitted from the last column on the plate. Compounds are seriallydiluted in dimethyl sulfoxide (DMSO, 99.7% pure, cell culture tested,Sigma-Aldrich, St. Louis, Mo., Catalog No. D2650) from 5 mM in 3-folddilutions and then diluted 1:250 in Yssel's medium. 50 μL of 2× compoundis added per well in duplicate and the cells are allowed to proliferatefor 72 hours at 37° C.

Proliferation is measured using CellTiter-Glo® Luminescent CellViability Assay (Promega), which determines the number of viable cellsin culture based on quantitation of the ATP present, as an indicator ofmetabolically active cells. The substrate is thawed and allowed to cometo room temperature. After mixing the Cell Titer-Glo reagent and diluenttogether, 100 μL is added to each well. The plates are mixed on anorbital shaker for two minutes to induce lysis and incubated at roomtemperature for an additional ten minutes to allow the signal toequilibrate. Detection is performed using a Wallac Victor2 1420multilabel counter purchased from Perkin Elmer, Shelton, Conn.

Example 346 A549 Epithelial Line Stimulated with IFNγ

A549 lung epithelial cells up-regulate ICAM-1 (CD54) surface expressionin response to a variety of different stimuli. Therefore, using ICAM-1expression as readout, compound effects on different signaling pathwayscan be assessed in the same cell type. IFNγ up-regulates ICAM-1 throughactivation of the JAK/Stat pathway. In this example, the up-regulationof ICAM-1 by IFNγ is assessed.

The A549 lung epithelial carcinoma cell line originated from theAmerican Type Culture Collection. Routine culturing is with F12K media(Mediatech Inc., Lenexa, Kans., Cat. No. 10-025-CV) with 10% fetalbovine serum, 100 I.U. penicillin and 100 ng/mL streptomycin (completeF12k media). Cells are incubated in a humidified atmosphere of 5% CO₂ at37° C. Prior to use in the assay, A549 cells are washed with PBS andtrypsinized (Mediatech Inc., Cat. No. 25-052-CI) to lift the cells. Thetrypsin cell suspension is neutralized with complete F12K media andcentrifuged to pellet the cells. The cell pellet is resuspended incomplete F12K media at a concentration of 2.0×10⁵/mL. Cells are seededat 20,000 per well, 100 μL total volume, in a flat bottom tissue cultureplate and allowed to adhere overnight.

On day two, A549 cells are pre-incubated with a 2,4-pyrimidinediaminetest compound or DMSO (control) (Sigma-Aldrich, St. Louis, Mo., CatalogNo. D2650) for 1 hour. The cells are then stimulated with IFNγ (75ng/mL) (Peprotech Inc., Rocky Hill, N.J., Cat. No. 300-02) and allowedto incubate for 24 hours. The final test compound dose range is 30 μM to14 nM in 200 μL F12K media containing 5% FBS, 0.3% DMSO.

On day three, the cell media is removed and the cells are washed with200 μL PBS (phosphate buffered saline). Each well is trypsinized todissociate the cells, then neutralized by addition of 200 μL completeF12K media. Cells are pelleted and stained with an APC conjugated mouseanti-human ICAM-1 (CD54) (BD Pharmingen, San Diego, Calif., Catalog#559771) antibody for 20 minutes at 4° C. Cells are washed with ice coldFACS buffer (PBS+2% FBS) and surface ICAM-1 expression is analyzed byflow cytometry. Detection is performed using a BD LSR I System FlowCytometer, purchased from BD Biosciences of San Jose, Calif. Events aregated for live scatter and the geometric mean is calculated(Becton-Dickinson CellQuest software version 3.3, Franklin Lakes, N.J.).Geometric means are plotted against the compound concentration togenerate a dose response curve.

Example 347 U937 IFNγICAM1 FACS Assay

U937 human monocytic cells up-regulate ICAM-1 (CD54) surface expressionin response to a variety of different stimuli. Therefore, using ICAM-1expression as readout, compound effects on different signaling pathwayscan be assessed in the same cell type. IFNγ up-regulates ICAM-1 throughactivation of the JAK/Stat pathway. In this example, the up-regulationof ICAM-1 by IFNγ is assessed.

The U937 human monocytic cell line is obtained from ATCC of Rockville,Md., catalog number CRL-1593.2, and cultured in RPMI-1640 mediumcontaining 10% (v/v) FCS. U937 cells are grown in 10% RPMI. The cellsare then plated at a concentration of 100,000 cells per 160 μL in 96well flat bottom plates. The test compounds are then diluted as follows:10 mM test compound is diluted 1:5 in DMSO (3 μL 10 mM test compound in12 μL DMSO), followed by a 1:3 serial dilution of test compound in DMSO(6 μL test compound serially diluted into 12 μL DMSO to give 3-folddilutions). Then 4 μL of test compound is transferred to 76 μL of 10%RPMI resulting in a 10× solution (100 μM test compound, 5% DMSO). Forcontrol wells, 4 μL of DMSO is diluted into 76 μL 10% RPMI.

The assay is performed in duplicate with 8 points (8 3-fold dilutionconcentrations from 10 μL) and with 4 wells of DMSO only (control wells)under stimulated conditions and 4 wells of DMSO only under unstimulatedconditions.

The diluted compound plate is mixed 2× using a multimek (Beckman Coulterof Brea, Calif.) and then 20 μL of the diluted compounds is transferredto the 96 well plate containing 160 μL of cells, which are then mixedagain twice at low speeds. The cells and compounds are thenpre-incubated for 30 minutes at 37° C. with 5% CO₂.

The 10× stimulation mix is made by preparing a 100 g/mL solution ofhuman IFNγ in 10% RPMI. The cells and compound are then stimulated with20 μL of IFNγ stimulation mix to give a final concentration of 10 ng/mLIFNγ, 10 μM test compound, and 0.5% DMSO. The cells are kept underconditions for stimulation for 18-24 hours at 37° C. with 5% CO₂.

The cells are transferred to a 96 well round bottom plate for stainingand then kept on ice for the duration of the staining procedure. Cellsare spun down at 1000 rpm for 5 minutes at 4° C., following which thesupernatant is removed. Following removal of the supernatant, 1 μL APCconjugated mouse anti-human ICAM-1 antibody is added per 100 μL FACSbuffer. The cells are then incubated on ice in the dark for 30 minutes.Following incubation, 150 μL of FACS buffer is added and the cells arecentrifuged at 1000 rpm for 5 minutes at 4° C., following which thesupernatant is removed. After removal of the supernatant, 200 μL of FACSbuffer is added and the cells are resuspended. After suspension, thecells are centrifuged at 1000 rpm for 5 min at 4° C. Supernatant is thenremoved prior to resuspension of the cells in 150 μL FACS buffer.

Detection is performed using a BD LSR I System Flow Cytometer, purchasedfrom BD Biosciences of San Jose, Calif. The live cells are gated forlive scatter and the geometric mean of ICAM-APC is measured(Becton-Dickinson CellQuest software version 3.3, Franklin Lakes, N.J.).Both % live cells and ICAM-1 expression is analyzed. The assays for thetest compounds is carried out in parallel with a control compound ofknown activity. The EC₅₀ for the control compound is typically 40-100nM.

Example 348 Analysis of B Cell Signaling

The human non-Hodgkin's lymphoma B cell lines SUDHL-4 (#ACC 495),SUDHL-6 (#ACC572), and Karpas-422 (#ACC32) were obtained from DSMZ(Braunschweig, Germany); Toledo (#CRL-2631) and Ramos (#CRL-1596) wereobtained from the American Type Culture Collection (ATCC; Manassas,Va.). All cells were maintained in RPMI media (Invitrogen, Carlsbad,Calif.) supplemented with 10% fetal calf serum (ATCC) andpenicillin/streptomycin (Invitrogen), and maintained in a 37° C.humidified tissue culture incubator. Antibodies used in these studiesinclude polyclonal goat F(ab)′2 anti-human IgG (H+L) and anti-human IgM(BioSource, Camarillo, Calif.); rabbit anti-human Syk, rabbit anti-humanphospho-Syk (Y525/526), rabbit anti-human phospho-Syk (Y352), anti-humanBLNK, anti-human phospho-BLNK (Y84) were obtained from Cell SignalingTechnologies, Inc. (Danvers, Mass.). The following antibodies wereobtained from Becton Dickenson (San Jose, Calif.) for phospho-flowcytometry: Alexa fluor 488-conjugated mouse anti-human phospho-STAT6(Y641), Phycoerythrin (PE)-conjugated mouse anti-human phospho-Zap70(Y319)/Syk(Y352), and Fluorescein isothiocyanate (FITC)-conjugated mouseanti-human phospho-ERK1/2 (T202/Y204).

Phospho-flow cytometry was performed essentially as described elsewhere(Irish, Czerwinski et al. Blood 108(9): 3135-42 (2006). 0.5×10⁶ cells ingrowth media were stimulated with 1 μg/ml anti-μ or anti-γ antibody for10 minutes. Induced signaling was terminated immediately following theindicated time by the addition of paraformaldehyde (Electron MicroscopySciences, Hatfield, Pa.) to a final concentration of 1%. Cells wereincubated with paraformaldehyde for 5 minutes at room temperature,washed once with phosphate buffered saline (PBS), then resuspended andincubated overnight at 4° C. in pre-chilled methanol (−80° C.) (company,address). Fixed and permeabalized cells were subsequently washed once inPBS, a second time in PBS containing 1% bovine serum albumin (BSA)(Sigma-Aldrich, St. Louis, Mo.), and then stained with the indicatedantibodies diluted 1:20 in PBS+1% BSA. After 30 minutes, cells werewashed once in PBS and subjected to flow cytometry using the FACSCalibur (Becton Dickenson). For Western blot analyses, 106 cells werestimulated for 30 minutes with 2 μg/ml of the indicated BCR-specificantibodies. Signaling was terminated by resuspending the cells in lysisbuffer and incubated on ice for 1 hour. Cell debris were removed bycentrifugation, and the cleared protein lysates were resolved by 10%SDS-PAGE and probed with the indicated antibodies followingrecommendations made by the manufacturers. Where indicated, cells werepre-treated for 1 hour at 37° C. with Syk inhibitors or vehicle control(0.5% DMSO) at several concentrations prior to stimulation with anti-BCRantibody.

Example 349 Selective Inhibition of Syk Activity

Compounds were tested for their ability to inhibit purified Syk. P459-72and P505-15 (two compounds from a Syk-specific series as shown in Table9b) and example 100b (from a series with dual Syk and JAK inhibitoryactivities) were found to suppress Syk kinase activity with IC50s of 43nM, 6 nM, and 31 nM, respectively. The selectivity of these compoundsfor Syk was determined by screening each against a panel of 270independent purified kinases at 300 nM (Millipore). The percentinhibition relative to vehicle control was calculated, and the numberswere converted into a heat-map; no inhibition is represented as green,increasing blending with red indicates increasing percent inhibitionwith yellow representing 50% inhibition and red representing 100%inhibition (FIG. 8). As depicted in FIG. 8A, P459-72 and P505-15 werehighly Syk specific (first and second rows, respectively) whereasexample 100b inhibited multiple kinases (third row). The subset ofkinases that were inhibited by ≧80% by any of the three compounds areshown in FIG. 8B. example 100b inhibited Syk and MLK-1 (first row). At300 nM P505-15 inhibited 10 different kinases (second row). Whenre-tested at 50 nM (approximately 10× above its Syk IC50 value of 6 nM),however, Syk was the only kinase that remained inhibited (third row).P420-89 inhibited Syk, JAK2 and JAK3, along with several other kinases(fourth row).

Employing the Milipore panel of purified kinases P505-15 (IC₅₀=1 nM)inhibited 98% of purified Syk kinase activity at 50 nM. IC50 values weredetermined for those kinases that were inhibited by >80% at 300 nM inthe Millipore kinase panel.

Kinase IC50 (nM) Syk(h) 1 MLK1 60 Fgr(h) 81 Yes(h) 123 Flt3(h) 139 PAK5166 Lyn(h) 199 cSRC(h) 244 Lck(h) 300

By contrast, multi-kinase inhibitor P420-89 is more akin to Rigel'sR788. At 300 nM, P420-89 inhibited Syk by 88%, along with >80%inhibition of 32 additional kinases. Among these were JAK 2 and 3 (93%and 85% inhibited, respectively), Flt-3 (83-92% inhibited), and cKit(95-97% inhibited), all targets for therapeutic manipulation oflymphocyte function.

Example 350 Calcium Flux Assay and Selective Inhibition of Syk inNon-Hodgkin's Lymphoma B Cell Lines

Ramos cells were cultured (maintaining approximately 0.5×106 cells/ml)in growth medium 3 to 4 days ahead of experiments. Cells were harvestedand re-suspended in fresh medium at 8×106 cells/ml before dye-loading.An equal volume of Calcium 3 loading dye (Molecular Device, Sunneyvale,Calif.) was added to the cell suspensions. Loaded cells were dispensedin a 96 well plate and incubated for 20 minutes. Syk inhibitors werethen added to the loaded cells and incubated for another 30 minutes. Bcells were stimulated with 5 μg/ml anti-μ antibody. Changes inintracellular Ca2+ concentration was measured using the FlexSTATion(Molecular Devices, Sunnyvale, Calif.).

The selectivity and potency of Syk inhibition in B cells was initiallyinterrogated by Western blot, measuring BCR-mediated induction of pSykY525/526 and pBLNK Y84, both measures of Syk kinase activity, and theinduction of pSyk Y352, a measure of Src kinase activity. SUDHL-6 Bcells were stimulated with anti-BCR specific antibody for 30 minutes inthe presence or absence of each Syk inhibitor or vehicle control.Treatment with 0.16 or 1 μM of each compound reduced BCR-induced Sykautophorphorylation (Y525/526) by roughly 40% and 60%, respectively, asestimated by densitometry (data not shown). An expanded range ofconcentrations was used to further evaluate the effect of thesecompounds on BCR induced Syk and Src kinase activity. As shown in FIG.9, A-C, each compound inhibited Syk activity (pBLNK Y84) with IC50values ranging from 0.16 to 1 μM, while no effect on Src activity (pSykY352) was observed as high as 2.5 μM.

The ability of each compound to suppress signaling events more distal tothe BCR was also measured. Cells were again stimulated by anti-BCRantibody in the presence or absence of various concentrations of eachSyk inhibitor. The induction of pSyk Y352 was measured as a specificitycontrol, while that of pERK1/2 T202/Y204 was used as a measure of moredistal Syk-dependent signaling (Jiang, Craxton et al. J Exp Med 188(7):1297-306 (1998). FIG. 12C shows representative FACS plots depicting theeffect of the most specific and potent Syk inhibitor of the three,P505-15, on BCR signaling. At 125 nM, ERK1/2 activation was completelysuppressed, whereas the stimulated cells still stained positive for SykY352. This experiment was repeated, in which the effect of all threecompounds on Src and Syk activity were determined (FIG. 10A).Concentrations of less than 125 nM were sufficient to suppress BCRinduced Syk signaling to ERK1/2. By contrast, much higher concentrationswere required to cause a modest suppression of Src activity; an effecton Src that was not observed by Western blot (FIG. 9, A-C). None ofthese Syk inhibitors suppressed PMA-induced ERK1/2 tyrosinephosphorylation, demonstrating these compounds do not inhibit signalingevents down-stream of PKC.

Whereas P459-72 and P505-15 specifically inhibited Syk in purified andcellular assays, example 100b additionally demonstrated activity againstpurified JAK kinases. These compounds were tested for inhibition of IL-4signaling to STAT-6 via JAK1/3 in B cells, a signaling pathway that doesnot require Syk. The Syk specific compounds did not suppress IL4signaling at concentrations as high as 2 μM. Conversely, example 100bdid suppress IL4 signaling, with an IC50 around 125 nM (FIG. 10B).

This shows that selective inhibition of Syk suppressed BCR-induced Ca2+flux in B cells with IC50 values around 100 nM. This suggests that byinhibiting Syk, these compounds suppress the signaling pathway, blockingthe cellular response.

Selective inhibition of Syk is sufficient to suppress BCR signalingwithout affecting Src (FIGS. 11 and 12) or JAK (FIG. 10B). Additionally,P505-15 and example 100b equally induced apoptosis in these cells (FIG.11B). This data demonstrates the role of Syk signaling in the survivalof NHL cell lines, and demonstrates that inhibition of kinases otherthan Syk is not required to achieve this effect.

Example 351 Caspase 3 and Proliferation Assays: Syk Inhibition DisruptsProliferation and Survival of Non-Hodgkin's Lymphoma B Cell Lines

Induction of apoptosis was measured using the PE-conjugated monoclonalactive caspase-3 antibody apoptosis kit (Becton Dickenson) following thesupplied protocol. Cells were suspended in growth media (0.5×106cells/ml) and treated with the indicated concentrations of each Sykinhibitor or vehicle control for 24, 48, or 72 hours prior to FACSanalysis. The MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide, a tetrazole) assay (company name) was used as a measure of cellviability and growth, following protocols supplied by the manufacturer.Cells were treated with the indicated concentrations of each Sykinhibitor or vehicle control for 72 hours.

SUDHL-4 and SUDHL-6 cells were previously classified as “BCR-type”(Monti, Savage et al. Blood 105(5): 1851-61 (2005); Polo, Juszczynski etal. Proc Natl Acad Sci USA 104(9): 3207-12 (2007) and sensitive to Sykinhibition by R406 (Chen, Monti et al. 2008). The Toledo and Karpas-422cell lines that lack BCR and BLNK expression, respectively (Gabay,Ben-Bassat et al. Eur J Haematol 63(3): 180-91 (1999); Sprangers,Feldhahn et al. Oncogene 25(36): 5056-62 (2006), having thereforeadapted to survive independent of BCR signals, were insensitive to R406(Chen, Monti et al. 2008). The proliferation of these cell lines whencultured in the presence or absence of various concentrations of eachSyk inhibitor for 72 hours was tested.

Selective inhibition of Syk was sufficient to induce apoptosis in“BCR-type” NHL cell lines. Cells were incubated with 1 or 3 μM of theinhibitor for 72 h. As demonstrated in FIG. 11A, SUDHL-4 and -6 cellseach underwent apoptosis, whereas the Toledo and Karpas-422 cells didnot (FIG. 11A). In replicate experiments, the specific inhibition of Sykby P459-72 and P505-15 induced apoptosis only in the SUDHL and Ramoscell lines. By comparison, example 100b, which potently inhibits Syk andJAK kinases, induced apoptosis in all the “BCR-type” cell lines, as wellas in Karpas-422 and JJN-3, a multiple myeloma cell line that lacks BCR,and BLNK expression (Sprangers, Feldhahn et al. Oncogene 25(36): 5056-62(2006). The Toledo cells remained insensitive to all three compounds(FIG. 11B). In a separate experiment, the SUDHL-6 and Toledo cells werefound to be equally sensitive to induction of apoptosis by 72 htreatment with 1 μM PMA. These data demonstrate the specific requirementof Syk in the survival of certain NHL cell lines.

Example 352 Xenograft Studies and Tumor and Plasma ConcentrationAnalysis

Syk Inhibition Protects Against Tumor Formation in a Xenograft MouseModel. Mice were received (company) and acclimated in-house at leastthree days prior to use. Ramos cells (3×106) were injectedsubcutaneously into the hind flank area of conscious mice using a 27gauge needle in an injection volume of less than 0.5 ml. Followinginjection, mice were randomized into treatment groups (n=15) and dosedtwice daily by oral gavage with vehicle or 10, 15, or 20 mg/kg of theinhibitor. Body weights were obtained at least once per week and calipermeasurements of tumors were determined twice per week beginning whenpalpable tumors were formed until the end of the study. Tumor volume wasassessed by caliper measurement using a formula [maximumlength×width×height×π/6]. Twice daily dosing of vehicle or the inhibitorcontinued until the vehicle or any treatment group exhibited tumors thatexceeded 1.5 grams in size. At the time of termination (5 weeks postRamos innoculation) the mice were anesthetized with a ketamine cocktail.A blood sample was obtained for CBC and plasma concentrationdetermination via cardiac puncture and the mice were euthanized viacervical dislocation. Tumors were then be excised and weighed. One halfof the tumor was snap frozen in liquid nitrogen for determination ofconcentration of the inhibitor in the tumor tissue and the other halfwas placed in 10% buffered formalin for histological investigation.

The effect of Syk inhibition on Ramos tumor formation in a xenograftmouse model was assessed. Mice were dosed twice daily with 10, 15, or 20mg/kg P505-15 or vehicle control beginning the day of tumor cellinoculation. Caliper measurements were initiated when tumors began toform, approximately three weeks post-tumor inoculation, and repeatedevery third day until termination of the study. The study was terminatedwhen tumor weights began reaching approximately 1.5 mg, at which timetumors were excised and weighed. Tumor and plasma samples were subjectedto pharmacokinetic analysis.

Each tumor sample was homogenized in 3 ml of saline per gram of tumorusing the Kontes® Microtube Pellet Pestle® Rods and Motor (Kimble Chase,Vineland, N.J.). Plasma and tumor samples were analyzed for P505-15concentration using a liquid chromatography tandem mass spectrometer(LC/MS/MS). In brief, plasma and tumor samples were processed in a96-well Captiva™ filter plate (0.2 μm, Varian, Inc., Palo Alto, Calif.).Aliquots of plasma and homogenized tumor samples were precipitated withacetonitrile containing 200 ng/mL of:

the internal standard. The mixture was vortexed and refrigerated at 4°C. for 30 minutes to allow complete protein precipitation. The mixturewas filtered into a 96-well collection plate. The filtrate was injectedonto a Sciex API3000 LC/MS/MS equipped with a turbo-ion spray source.P505-15 and Compound A were separated on a Phenomenex Luna 5μ HILICcolumn (4.6×100 mm, 5 mm; Phenomenex, Torrance, Calif.). A mobile phasegradient mixture of 10% mobile phase A (0.1% formic acid in water) and90% mobile phase B (0.1% formic acid in 90% acetonitrile, 10% water) to65% mobile phase B was programmed over 1.1 minutes followed by agradient of mobile phase B from 65% to 90% over 0.01 minutes. The peakareas of the m/z 394/360 product ion of P505-15 were measured againstthose of the m/z 357/295 product ion of Compound A (internal standard)in positive ion mode. The analytical range was 2 to 5000 ng/ml.

Pharmacokinetic analysis revealed that at steady-state, tumorconcentrations of P505-15 followed the concentration-time profiles seenwith plasma in the 10, 15, and 20 mg/kg dose groups. Nonlinear increasesin Cmax, AUC (0-8), and tumor Cmin were observed as the dose wasincreased, but a dose-proportional increase in plasma Cmin was noted.Mean Cmax and AUC (0-8) in plasma was at least 2-fold greater than thatin tumor for all doses examined; however, mean nadir concentrations(Cmin) were higher in tumor than in plasma (Table 11A), indicatingaccumulation of P505-15 in the tumor compartment.

TABLE 11A Tmax Cmin Cmax AUC (0-8) Dosing regiment (hr) (ng/mL) (ng/mL)(ng * hr/mL) Determined from plasma 10 mg/kg BID 1.50 17.6 179 738 15mg/kg BID 1.50 26.6 343 1671 20 mg/kg BID 4.00 39.5 570 3191 Determinedfrom tumor 10 mg/kg BID 8.00 24.5 55.2 353 15 mg/kg BID* 4.00 67.8 163475 20 mg/kg BID 4.00 125 252 1453

TABLE 11B tumor/plasma ratio Dosing regimen AUC based Cmax based Cminbased 10 mg/kg BID 0.478 0.308 1.39 15 mg/kg BID* 0.284 0.475 2.55 20mg/kg BID 0.455 0.442 3.15 Note: Nadir (0), 1.5, 4, and 8 h samples weretaken on the da of harvest following the AM dose. The second dose wasnot administered on the day of harvest; therefore, pharmacokineticvalues above were determined after a single AM dose at steady-state.*Only one tumor sample was available for the 8 h time-point and may havebeen an outlier (tumor concentrations at 8 h - 608 ng/ml); therefore,pharmacokinetic parameters were determined between 0 to 4 h for the 15mg/kg BID P505-15 dose group. As a result, AUC (0-8) and AUC basedtumor/plamsa ration for this dose group may be underestimated.

The difference between plasma and tumor Cmin became more prominent asthe dose was increased, as indicated by the increase in tumor/plasmaratios determined from Cmin (Table 11B). Tumor/plasma ratios determinedfrom Cmax and AUC (0-8) were similar across the various dose groups.Tumor concentrations were sustained above 60, 170, and 640 nM over theentire dosing interval at steady-state for P505-15 at 10, 15, and 20mg/kg, respectively.

Mice dosed with all three concentrations of P505-15 were protected fromRamos tumor growth in vivo. This was first evident from calipermeasurements (data not shown), which revealed a reduced rate of tumorgrowth in the presence of the Syk inhibitor. Upon study completion, micewere euthanized and tumors excised and weighed. Consistent with calipermeasurements, a statistically significant reduction in average tumorweight was achieved in all dosing groups, relative to vehicle control.

The Syk-specific inhibitor P505-15 was also tested for activity in aRamos tumor mouse xenograft model. At all the concentrations tested,statistically significant reductions in tumor growth were observed inmice dosed BID with P505-15. The lowest concentration tested was 10mg/kg, achieving tumor concentrations ranging from 64 to 140 nM over thecourse of the day. Suppression of tumor growth at these concentrationsin vivo is consistent with concentrations of <125 nM found to suppressBCR-induced Ca2+ flux and distal BCR signaling to pERK Y204 (FIG. 12).The selective pharmacological inhibition of Syk results in effects onthe proliferations and survival of NHL cell lines. These data suggestthat the selective targeting of Syk may similarly have clinical benefitin a variety of B-cell proliferative disorders.

As detailed herein, Syk has been implicated experimentally in B celldevelopment, proliferation, and survival. Moreover, Syk is implicated asan oncogene. Expression of constitutively active Syk in adoptivelytransferred bone marrow cells induces leukemia in mice, andover-activity of Syk is associated with a variety of lymphomas in humansGiven the role of Syk in B cell biology, its selective inhibition may besufficient to provide clinical benefit in B cell proliferativedisorders, while reducing toxicities that may arise due to suppressionof other off-target kinases.

The present invention provides a number of embodiments. It is apparentthat the examples may be altered to provide other embodiments of thisinvention. Therefore, it will be appreciated that the scope of thisinvention is to be defined by the appended claims rather than by thespecific embodiments, which have been represented by way of example.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety. From the foregoing it will be appreciatedthat, although specific embodiments of the invention have been describedherein for purposes of illustration, various modifications may be madewithout deviating from the spirit and scope of the invention.Accordingly, the invention is not limited except as by the appendedclaims.

What is claimed is:
 1. A compound having the formula I:

or a pharmaceutically acceptable salt thereof, wherein: D¹ isC₃₋₈cycloalkyl, optionally substituted with from 1 to 4 substituentsindependently selected from the group consisting of: C₁₋₈ alkyl, amino,hydroxy, C₁₋₈alkylcarbonyl, aminocarbonyl, C₁₋₈alkoxycarbonylamino,arylC₁₋₈alkoxycarbonylamino, phenyl and heterocyclylC₁₋₈alkylene;wherein the cycloalkyl does not have more than one double bond; R¹ isselected from the group consisting of H, C₁₋₈ alkyl, amino,aminocarbonyl, hydroxyl, C₁₋₈ alkoxy, C₁₋₈ haloalkyl, C₂₋₈ alkenyl, C₂₋₈alkynyl, oxo, cyano, C₁₋₈ alkoxycarbonyl, C₃₋₈ cycloalkyl, aryl andheterocyclyl; and each heterocyclyl is optionally substituted with from1 to 4 substituents selected from the group consisting of: C₁₋₈ alkyl,halo, oxo, amino, C₁₋₈alkoxy, C₁₋₈alkylcarbonyl, arylC₁₋₈alkoxycarbonyl, aminocarbonyl, arylC₁₋₈ alkylenecarbonyl and C₁₋₈alkylsulfonyl Y¹ is aryl; optionally substituted with from 1 to 3substituents, R^(4a), independently selected from the group consistingof C₁₋₈alkyl, C₁₋₈alkoxyC₁₋₈alkyl, aminocarbonyl-, hydroxyl, oxo,halogen, hydroxy, C₁₋₈alkoxy and C₁₋₈alkylsulfonyl; R² is selected fromthe group consisting of heterocyclyl and heteroaryl, each of which issubstituted with at least one group, R³, selected from the groupconsisting of aminoC₁₋₈alkyl-, C₁₋₈alkoxyC₁₋₈alkyl-, oxo-,C₁₋₈alkylcarbonyl, C₃₋₈cycloalkylcarbonyl, heterocyclylcarbonyl,C₁₋₈alkylcarbonylamino, C₃₋₈cycloalkylcarbonylamino,heterocyclylcarbonylamino, aminosulfonyl, C₁₋₈alkylsulfonyl,C₃₋₈cycloalkylsulfonyl and heterocyclylsulfonyl; and wherein R² isfurther optionally substituted with from 1 to 2 substituents, R^(4c),independently selected from the group consisting of C₁₋₈ alkyl,C₁₋₈alkoxy, halo, aminocarbonyl, oxo, hydroxyl, aminoC₁₋₈alkylene,C₁₋₈alkoxyC₁₋₈alkylene, C₁₋₈alkylcarbonyl, C₃₋₈cycloalkylcarbonyl,heterocyclylcarbonyl, C₁₋₈alkylcarbonylamino,C₃₋₈cycloalkylcarbonylamino, heterocyclylcarbonylamino,C₁₋₈alkylsulfonyl, C₃₋₈cycloalkylsulfonyl, heterocyclylsulfonyl,C₃₋₈cycloalkyl, C₁₋₈alkylcycloalkylene, and heteroaryl.
 2. The compoundof claim 1, wherein: Y¹ is phenyl.
 3. The compound of claim 1, wherein:R² is heterocyclyl.
 4. The compound of claim 1, wherein the moiety—Y¹—R² is selected from the group consisting of:

Y² is CH or C; R^(4a) is selected from the group consisting of H andC₁₋₈alkyl; each R^(4c) is independently selected from the groupconsisting of H, C₁₋₈alkyl, C₁₋₈alkoxyC₁₋₈alkyl, aminocarbonyl-,hydroxyl, oxo, C₁₋₈alkoxy and halo; each R^(4b) is independentlyselected from the group consisting of H, C₁₋₈alkyl, amino, C₁₋₈alkoxyand heterocyclyl; the subscript p is 0, 1, or 2; and the wavy lineindicates the point of attachment to the rest of the molecule.
 5. Thecompound of claim 4, wherein —Y¹—R² is selected from the groupconsisting of:

Y² is C; each R^(4a) is selected from the group consisting of H andhalo; each R^(4c) is independently selected from the group consisting ofH and C₁₋₈alkyl; each R^(4b) is independently selected from the groupconsisting of H and C₁₋₈alkyl; the subscript p is 0, 1, or 2; and thewavy line indicates the point of attachment to the rest of the molecule.6. The compound of claim 4, wherein the moiety —Y¹—R² is selected fromthe group consisting of:

and the wavy line indicates the point of attachment to the rest of themolecule.
 7. The compound of claim 6, having the formula Id:

or a pharmaceutically acceptable tautomer, salt, or stereoisomerthereof.
 8. The compound of claim 6, having the formula Id1:

or a pharmaceutically acceptable tautomer, salt, or stereoisomerthereof.
 9. The compound of claim 6, having the formula:

or a pharmaceutically acceptable tautomer, salt, or stereoisomerthereof.
 10. The compound of claim 1, wherein: D¹ is cyclopropyl. 11.The compound of claim 1, wherein: D¹ is cyclobutyl.
 12. The compound ofclaim 1, wherein: D¹ is cyclopentyl.
 13. The compound of claim 1,wherein: D¹ is cyclohexyl.
 14. The compound of claim 1, wherein themoiety —Y¹—R² is selected from the group consisting of:

Y² is C; each R^(4a) is selected from the group consisting of H andC₁₋₈alkyl; each R^(4c) is independently selected from the groupconsisting of H, C₁₋₈alkyl, C₁₋₈alkoxyC₁₋₈alkyl, aminocarbonyl-,hydroxyl, oxo, C₁₋₈alkoxy and halo; each R^(4b) is independentlyselected from the group consisting of H, C₁₋₈alkyl, amino, C₁₋₈alkoxyand heterocyclyl; and the subscript p is 0, 1, or 2; and the wavy lineindicates the point of attachment to the rest of the molecule.
 15. Acompound having the formula:

or a pharmaceutically acceptable tautomer, salt, or stereoisomerthereof.
 16. A compound having the formula:

a pharmaceutically acceptable tautomer, salt, or stereoisomer thereof.17. A compound having the formula:

or a pharmaceutically acceptable tautomer, salt, or stereoisomerthereof.
 18. A composition comprising a compound of any one of thepreceding claims, in combination with a pharmaceutically acceptablecarrier or diluent.
 19. A kit comprising a composition of claim 18,packaging and instructions for use.